Antibodies to notch receptors

ABSTRACT

The present invention relates to compositions and methods for characterizing, diagnosing, and treating cancer. In particular the invention provides the means and methods for the diagnosis, characterization, prognosis and treatment of cancer and specifically targeting cancer stem cells. The present invention provides an antibody that specifically binds to a non-ligand binding region of the extracellular domain of a human NOTCH receptor and inhibits growth of tumor cells. The present invention further provides a method of treating cancer, the method comprising administering a therapeutically effective amount of an antibody that specifically binds to a non-ligand binding region of the extracellular domain of a human NOTCH receptor protein and inhibits growth of tumor cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Prov. Appl. No. 60/812,955,filed Jun. 13, 2006; U.S. Prov. Appl. No. 60/879,336, filed Jan. 9,2007; and U.S. Prov. Appl. No. 60/878,661, filed Jan. 5, 2007; each ofwhich are herein incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to compositions and methods forcharacterizing, diagnosing, and treating cancer. In particular, theinvention provides the means and methods for diagnosis,characterization, prognosis, and treatment of cancer and specificallytargeting cancer stem cells. The present invention provides an antibodythat specifically binds to a non-ligand binding region of theextracellular domain of a human NOTCH receptor and inhibits growth oftumor cells. The present invention further provides a method of treatingcancer, the method comprising administering a therapeutically effectiveamount of an antibody that specifically binds to a non-ligand bindingregion of the extracellular domain of a human NOTCH receptor protein andinhibits growth of tumor cells.

2. Background Art

Cancer is one of the leading causes of death in the developed world,resulting in over 500,000 deaths per year in the United States alone.Over one million people are diagnosed with cancer in the U.S. each year,and overall it is estimated that more than 1 in 3 people will developsome form of cancer during their lifetime. Though there are more than200 different types of cancer, four of them—breast, lung, colorectal,and prostate—account for over half of all new cases (Jemal et al.,Cancer J. Clin. 53:5-26 (2003)).

Breast cancer is the most common cancer in woman, with an estimate 12%of women at risk of developing the disease during their lifetime.Although mortality rates have decreased due to earlier detection andimproved treatments, breast cancer remains a leading cause of death inmiddle-aged women. Furthermore, metastatic breast cancer is still anincurable disease. On presentation, most patients with metastatic breastcancer have only one or two organ systems affected, but as the diseaseprogresses, multiple sites usually become involved. The most commonsites of metastatic involvement are locoregional recurrences in the skinand soft tissues of the chest wall, as well as in axilla andsupraclavicular areas. The most common site for distant metastasis isthe bone (30 40% of distant metastasis), followed by the lungs andliver. And although only approximately 1-5% of women with newlydiagnosed breast cancer have distant metastasis at the time ofdiagnosis, approximately 50% of patients with local disease eventuallyrelapse with metastasis within five years. At present the mediansurvival from the manifestation of distant metastases is about threeyears.

Current methods of diagnosing and staging breast cancer include thetumor-node-metastasis (TNM) system that relies on tumor size, tumorpresence in lymph nodes, and the presence of distant metastases asdescribed in the American Joint Committee on Cancer: AJCC Cancer StagingManual. Philadelphia, Pa.: Lippincott-Raven Publishers, 5th ed., 1997,pp 171-180, and in Harris, J R: “Staging of breast carcinoma” in Harris,J. R., Hellman, S., Henderson, I. C., Kinne D. W. (eds.): BreastDiseases. Philadelphia, Lippincott, 1991. These parameters are used toprovide a prognosis and select an appropriate therapy. The morphologicappearance of the tumor may also be assessed but because tumors withsimilar histopathologic appearance can exhibit significant clinicalvariability, this approach has serious limitations. Finally assays forcell surface markers can be used to divide certain tumor types intosubclasses. For example, one factor considered in the prognosis andtreatment of breast cancer is the presence of the estrogen receptor (ER)as ER-positive breast cancers typically respond more readily to hormonaltherapies such as tamoxifen or aromatase inhibitors than ER-negativetumors. Yet these analyses, though useful, are only partially predictiveof the clinical behavior of breast tumors, and there is much phenotypicdiversity present in breast cancers that current diagnostic tools failto detect and current therapies fail to treat.

Prostate cancer is the most common cancer in men in the developed world,representing an estimated 33% of all new cancer cases in the U.S., andis the second most frequent cause of death (Jemal et al., 2003, CACancer J. Clin. 53:5-26). Since the introduction of the prostatespecific antigen (PSA) blood test, early detection of prostate cancerhas dramatically improved survival rates, and the five year survivalrate for patients with local and regional stage prostate cancers at thetime of diagnosis is nearing 100%. Yet more than 50% of patients willeventually develop locally advanced or metastatic disease(Muthuramalingam et al., 2004, Clin. Oncol. 16:505-16).

Currently radical prostatectomy and radiation therapy provide curativetreatment for the majority of localized prostate tumors. However,therapeutic options are very limited for advanced cases. For metastaticdisease, androgen ablation with luteinising hormone-releasing hormone(LHRH) agonist alone or in combination with anti-androgens is thestandard treatment. Yet despite maximal androgen blockage, the diseasenearly always progresses with the majority developingandrogen-independent disease. At present there is no uniformly acceptedtreatment for hormone refractory prostate cancer, and chemotherapeuticregimes are commonly used (Muthuramalingam et al., 2004, Clin. Oncol.165:505-16; Trojan et al., 2005, Anticancer Res. 25:551-61).

Colorectal cancer is the third most common cancer and the fourth mostfrequent cause of cancer deaths worldwide (Weitz et al., 2005, Lancet365:153-65). Approximately 5-10% of all colorectal cancers arehereditary with one of the main forms being familial adenomatouspolyposis (FAP), an autosomal dominant disease in which about 80% ofaffected individuals contain a germline mutation in the adenomatouspolyposis coli (APC) gene. Colorectal carcinoma has a tendency to invadelocally by circumferential growth and elsewhere by lymphatic,hematogenous, transperitoneal, and perineural spread. The most commonsite of extralymphatic involvement is the liver, with the lungs the mostfrequently affected extra-abdominal organ. Other sites of hematogenousspread include the bones, kidneys, adrenal glands, and brain.

The current staging system for colorectal cancer is based on the degreeof tumor penetration through the bowel wall and the presence or absenceof nodal involvement. This staging system is defined by three majorDuke's classifications: Duke's A disease is confined to submucosa layersof colon or rectum; Duke's B disease has tumors that invade through themuscularis propria and may penetrate the wall of the colon or rectum;and Duke's C disease includes any degree of bowel wall invasion withregional lymph node metastasis. While surgical resection is highlyeffective for early stage colorectal cancers, providing cure rates of95% in Duke's A patients, the rate is reduced to 75% in Duke's Bpatients and the presence of positive lymph node in Duke's C diseasepredicts a 60% likelihood of recurrence within five years. Treatment ofDuke's C patients with a post surgical course of chemotherapy reducesthe recurrence rate to 40%-50%, and is now the standard of care forthese patients.

Lung cancer is the most common cancer worldwide, the third most commonlydiagnosed cancer in the United States, and by far the most frequentcause of cancer deaths (Spiro et al., 2002, Am. J. Respir. Crit. CareMed. 166:1166-96; Jemal et al., 2003, CA Cancer J. Clin. 53:5-26).Cigarette smoking is believed responsible for an estimated 87% of alllung cancers, making it the most deadly preventable disease. Lung canceris divided into two major types that account for over 90% of all lungcancers: small cell lung cancer (SCLC) and non-small cell lung cancer(NSCLC). SCLC accounts for 15-20% of cases and is characterized by itsorigin in large central airways and histological composition of sheetsof small cells with little cytoplasm. SCLC is more aggressive thanNSCLC, growing rapidly and metastasizing early and often. NSCLC accountsfor 80-85% of all cases and is further divided into three major subtypesbased on histology: adenocarcinoma, squamous cell carcinoma (epidermoidcarcinoma), and large cell undifferentiated carcinoma.

Lung cancer typically presents late in its course, and thus has a mediansurvival of only 6-12 months after diagnosis and an overall 5 yearsurvival rate of only 5-10%. Although surgery offers the best chance ofa cure, only a small fraction of lung cancer patients are eligible withthe majority relying on chemotherapy and radiotherapy. Despite attemptsto manipulate the timing and dose intensity of these therapies, survivalrates have increased little over the last 15 years (Spiro et al., 2002,Am. J. Respir. Crit. Care Med. 166:1166-96).

Cancer arises from dysregulation of the mechanisms that control normaltissue development and maintenance, and increasingly stem cells arethought to play a central role (Bleachy et al., 2004, Nature 432:324).During normal animal development, cells of most or all tissues arederived from normal precursors, called stem cells (Morrison et al.,1997, Cell 88:287-98; Morrison et al., 1997, Curr. Opin. Immunol.9:216-21; Morrison et al., 1995, Annu. Rev. Cell. Dev. Biol. 11:35-71).Stem cells are cell that: (1) have extensive proliferative capacity; 2)are capable of asymmetric cell division to generate one or more kinds ofprogeny with reduced proliferative and/or developmental potential; and(3) are capable of symmetric cell divisions for self-renewal orself-maintenance. The best-known example of adult cell renewal by thedifferentiation of stem cells is the hematopoietic system wheredevelopmentally immature precursors (hematopoietic stem and progenitorcells) respond to molecular signals to form the varied blood andlymphoid cell types. Other cells, including cells of the gut, breastductal system, and skin are constantly replenished from a smallpopulation of stem cells in each tissue, and recent studies suggest thatmost other adult tissues also harbor stem cells, including the brain.

Solid tumors are composed of heterogeneous cell populations. Forexample, breast cancers are a mixture of cancer cells and normal cells,including mesenchymal (stromal) cells, inflammatory cells, andendothelial cells. Classic models of cancer hold that phenotypicallydistinct cancer cell populations all have the capacity to proliferateand give rise to a new tumor. In the classical model, tumor cellheterogeneity results from environmental factors as well as ongoingmutations within cancer cells resulting in a diverse population oftumorigenic cells. This model rests on the idea that all populations oftumor cells would have some degree of tumorigenic potential. (Pandis etal., 1998, Genes, Chromosomes & Cancer 12:122-129; Kuukasjrvi et al.,1997, Cancer Res. 57:1597-1604; Bonsing et al., 1993, Cancer 71:382-391;Bonsing et al., 2000, Genes Chromosomes & Cancer 82: 173-183; Beerman Het al., 1991, Cytometry. 12:147-54; Aubele M & Werner M, 1999, Analyt.Cell. Path. 19:53; Shen L et al., 2000, Cancer Res. 60:3884).

An alternative model for the observed solid tumor cell heterogeneity isthat solid tumors result from a “solid tumor stem cell” (or “cancer stemcell” from a solid tumor) that subsequently undergoes chaoticdevelopment through both symmetric and asymmetric rounds of celldivisions. In this stem cell model, solid tumors contain a distinct andlimited (possibly even rare) subset of cells that share the propertiesof normal “stem cells”, in that they extensively proliferate andefficiently give rise both to additional solid tumor stem cells(self-renewal) and to the majority of tumor cells of a solid tumor thatlack tumorigenic potential. Indeed, mutations within a long-lived stemcell population may initiate the formation of cancer stem cells thatunderlie the growth and maintenance of tumors and whose presencecontributes to the failure of current therapeutic approaches.

The stem cell nature of cancer was first revealed in the blood cancer,acute myeloid leukemia (AML) (Lapidot et al., 1994, Nature 17:645-8).More recently it has been demonstrated that malignant human breasttumors similarly harbor a small, distinct population of cancer stemcells enriched for the ability to form tumors in immunodeficient mice.An ESA+, CD44+, CD24−/low, Lin− cell population was found to be 50-foldenriched for tumorigenic cells compared to unfractionated tumor cells(Al-Hajj et al., 2003, PNAS 100:3983-8). The ability to prospectivelyisolate the tumorigenic cancer cells has permitted investigation ofcritical biological pathways that underlie tumorigenicity in thesecells, and thus promises the development of better diagnostic assays andtherapeutics for cancer patients. It is toward this purpose that thisinvention is directed.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a method of treating cancer, wherein thecancer comprises cancer stem cells, comprising administering to thesubject a therapeutically effective amount of an antibody which binds acancer stem cell marker. In a more particular aspect, the presentinvention provides a method of treating cancer, wherein the cancercomprises stem cells expressing one or more Notch receptor familymembers, comprising administering to the subject a therapeuticallyeffective amount of an antibody that binds those Notch receptor familymembers or the ligands to those Notch receptors. The present inventionfor the first time identifies antibodies that bind to the non-ligandbinding domain of the extracellular domain of a human NOTCH receptor astherapeutically effective against cancer. Thus in certain embodimentsthe present invention provides an antibody that specifically binds to anon-ligand binding region of the extracellular domain of a human NOTCHreceptor and inhibits growth of tumor cells. In certain embodiments, thepresent invention further provides a method of treating cancer, themethod comprising administering a therapeutically effective amount of anantibody that specifically binds to a non-ligand binding region of theextracellular domain of a human NOTCH receptor protein and inhibitsgrowth of tumor cells.

Various advantages in using an antibody that binds Notch receptor familymembers or the ligands to those Notch receptors to treat such cancer arecontemplated herein. In particular, certain Notch receptors are highlyexpressed in certain solid tumors, for example, breast and colon, andthis provides a sink for active drug where the drug binds to the Notchreceptor. Antibodies that bind overexpressed Notch receptors areanticipated to have a better safety profile than currently availablechemotherapeutic drugs.

The invention further provides a method of treating cancer in a human,wherein the cancer comprising cancer stem cells is not characterized byoverexpression by the cancer stein cell of one or more Notch receptors,comprising administering to the human a therapeutically effective amountof an antibody which binds to a Notch receptor and blocks ligandactivation of a Notch receptor.

The invention further provides a method of treating cancer in a humancomprising administering to the human therapeutically effective amountsof (a) a first antibody which binds a Notch receptor and inhibits growthof cancer stem cells which overexpress Notch receptors; and (b) a secondantibody which binds a Notch receptor and blocks ligand activation of aNotch receptor.

The invention also provides a method of treating cancer, wherein thecancer is selected from the group consisting of breast, colon, rectaland colorectal cancer, comprising administering a therapeuticallyeffective amount of an antibody which binds Notch. The invention alsoprovides another method of treating cancer, wherein the cancer isselected from the group consisting of breast, colon, pancreatic,prostate, lung, rectal and colorectal cancer, comprising administering atherapeutically effective amount of an antibody that blocks ligandactivation of a Notch receptor. The invention also provides stillanother method of treating cancer, wherein the cancer is selected fromthe group consisting of breast, colon, pancreatic, prostate, lung,rectal and colorectal cancer, comprising administering a therapeuticallyeffective amount of an antibody that binds Notch and an antibody thatblocks ligand activation of a Notch receptor.

In further embodiments, the invention provides articles of manufacturefor use (among other things) in the above methods. For example, theinvention provides an article of manufacture comprising a container anda composition contained therein, wherein the composition comprises anantibody that binds Notch, and further comprises a package insertindicating that the composition can be used to treat cancer comprisingcancer stem cells. Another example, the invention provides an article ofmanufacture comprising a container and a composition contained therein,wherein the composition comprises an antibody that binds Notch, andfurther comprises a package insert indicating that the composition canbe used to treat cancer comprising cancer stem cells that express one ormore Notch receptors.

The invention additionally pertains to an article of manufacturecomprising a container and a composition contained therein, wherein thecomposition comprises an antibody which binds a Notch receptor andblocks ligand activation of a Notch receptor, and further comprises apackage insert indicating that the composition can be used to treatcancer, wherein the cancer comprises cancer stem cells that are notcharacterized by overexpression of the Notch receptor.

In certain embodiments, an article of manufacture is provided whichcomprises (a) a first container with a composition contained therein,wherein the composition comprises a first antibody that binds a Notchreceptor and inhibits growth of cancer cells comprising cancer stemcells overexpressing Notch; and (b) a second container with acomposition contained therein, wherein the composition comprises asecond antibody which binds Notch and blocks ligand activation of aNotch receptor.

A further article of manufacture is provided which comprises a containerand a composition contained therein, wherein the composition comprisesan antibody which binds Notch and blocks ligand activation of a Notchreceptor, and further comprises a package insert indicating that thecomposition can be used to treat a cancer selected from the groupconsisting of colon, pancreatic, prostate, lung, rectal and colorectalcancer.

The invention additionally provides: a humanized antibody which bindsNotch and blocks ligand activation of a Notch receptor; a compositioncomprising the humanized antibody and a pharmaceutically acceptablecarrier; and an immunoconjugate comprising the humanized antibodyconjugated with a cytotoxic agent.

Moreover, the invention provides isolated nucleic acid encoding thehumanized antibody; a vector comprising the nucleic acid; a host cellcomprising the nucleic acid or the vector; as well as a process ofproducing the humanized antibody comprising culturing a host cellcomprising the nucleic acid so that the nucleic acid is expressed and,optionally, further comprising recovering the humanized antibody fromthe host cell culture (e.g. from the host cell culture medium).

The invention further pertains to an immunoconjugate comprising anantibody that binds Notch conjugated to one or more calicheamicinmolecules, and the use of such conjugates for treating Notch expressingcancer, e.g., a cancer in which cancer stem cells overexpress Notch.

In another aspect, the present invention provides a method of treatingcancer, wherein the cancer comprises cancer stem cells, comprisingadministering to the subject a therapeutically effective amount of areceptor fusion protein which binds a ligand to a cancer stem cellmarker. In a more particular aspect, the present invention provides amethod of treating cancer, wherein the cancer comprises stem cellsoverexpressing one or more Notch receptor family members, comprisingadministering to the subject a therapeutically effective amount of areceptor fusion protein that binds one or more ligands to those Notchreceptor family members.

The invention further provides a method of treating cancer in a human,wherein the cancer comprising cancer stem cells is not characterized byoverexpression by the cancer stem cell of one or more Notch receptors,comprising administering to the human a therapeutically effective amountof a receptor fusion protein which binds to one or more ligands to Notchreceptor.

The invention further provides a method of treating cancer in a humancomprising administering to the human therapeutically effective amountsof (a) a receptor fusion protein which binds ligand to a Notch receptorand blocks ligand activation of a Notch receptor; and (b) an antibodywhich binds a Notch receptor and inhibits growth of cancer stem cellswhich overexpress Notch receptors.

The invention also provides a method of treating cancer, wherein thecancer is selected from the group consisting of breast, colon,pancreatic, rectal and colorectal cancer, comprising administering atherapeutically effective amount of a receptor fusion protein whichbinds one or more ligands to a Notch receptor. The invention alsoprovides still another method of treating cancer, wherein the cancer isselected from the group consisting of breast, colon, pancreatic, rectaland colorectal cancer, comprising administering a therapeuticallyeffective amount of a receptor fusion protein that blocks ligandactivation of a Notch receptor and an antibody that binds a Notchreceptor and inhibits growth of cancer stem cells which overexpressNotch receptors.

In further embodiments, the invention provides articles of manufacturefor use (among other things) in the above methods. For example, theinvention provides an article of manufacture comprising a container anda composition contained therein, wherein the composition comprises areceptor fusion protein that blocks ligand activation of a Notchreceptor, and further comprising a package insert indicating that thecomposition can be used to treat cancer comprising cancer stem cells.Another example, the invention provides an article of manufacturecomprising a container and a composition contained therein, wherein thecomposition comprises a receptor fusion protein that blocks ligandactivation of a Notch receptor, and further comprising a package insertindicating that the composition can be used to treat cancer comprisingcancer stem cells that express one or more Notch receptors.

The invention additionally pertains to an article of manufacturecomprising a container and a composition contained therein, wherein thecomposition comprises a receptor fusion protein which blocks ligandactivation of a Notch receptor, and further comprises a package insertindicating that the composition can be used to treat cancer, wherein thecancer comprises cancer stem cells that are not characterized byoverexpression of the Notch receptor.

In a further embodiment, an article of manufacture is provided whichcomprises (a) a first container with a composition contained therein,wherein the composition comprises a receptor fusion protein which blocksligand activation of a Notch receptor; and (b) a second container with acomposition contained therein, wherein the composition comprises anantibody that binds a Notch receptor and inhibits growth of cancer cellscomprising cancer stem cells overexpressing Notch.

A further article of manufacture is provided which comprises a containerand a composition contained therein, wherein the composition comprises areceptor fusion protein that blocks ligand activation of a Notchreceptor, and further comprises a package insert indicating that thecomposition can be used to treat a cancer selected from the groupconsisting of colon, pancreatic, prostate, lung, rectal and colorectalcancer.

Examples of solid tumors that can be treated using a therapeuticcomposition of the instant invention, for example, an antibody thatbinds Notch or a receptor fusion protein that blocks ligand activationof a Notch receptor include, but are not limited to, sarcomas andcarcinomas such as, but not limited to: fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma. The invention is applicable to sarcomas and epithelialcancers, such as ovarian cancers and breast cancers.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1: Epitope mapping of anti-NOTCH1 monoclonal antibodies that bindto non-ligand binding domains. Fc fusion proteins containing a deletionseries of NOTCH1 EGF domains 1-5 (A, B) or 10-15 (C) were separated bySDS-PAGE and blotted with monoclonal antibody 13M57 (A, B) or 31M80 (C).In contrast to anti-Fc antibodies that detected fusion proteins in alllanes, antibody 13M57 only detected fusion proteins containing EGFrepeat 4 including EGF 1-4 and EGF 1-5 but not EGF 1-3 (A); and EGF 4-5but not EGF 5 alone (B). Similarly, antibody 31M80 only detected fusionproteins containing EGF repeat 13 (C).

FIG. 2: Binding of anti-NOTCH1 monoclonal antibody 13M57 and 31M80 tocell surface expressed NOTCH1. (A) FACS analysis of cells co-expressingfull length NOTCH1 receptor and GFP incubated with, from left to right,IgG1 control antibodies, 13M57 and anti-human NOTCH1 EGF 1-5 antisera.(B) FAC analysis of mock transfected cells or cells expressing fulllength NOTCH1 receptor incubated with anti-NOTCH1 receptor antibody31M80 demonstrates that 31M80 specifically recognizes NOTCH1 receptor.Antibody binding relative to an IgG control antibody is inhibited byincreasing amounts of antigen protein containing EGF repeats 10-16linked to human Fc (0.5×, 3×, and 10× Ag31) but not antigen proteincontaining EGF repeats 1-5 linked to Fc (0.5×, 3×, and 10× Ag13). (C)Anti-NOTCH1 antibodies 13M57 and 31M80 showed increased binding to PE13breast tumor cells compared to an isotype control antibody (bottom) andthis binding corresponded to cells that express high levels of ESA andCD44 (top: CD44 x-axis and NOTCH1 y-axis). Cells isolated for atumorigenicity study are indicated in the flow cytometry results forantibody 31M80. (D) Anti-NOTCH1 antibodies 13M57 and 31M80 showedincreased binding to T3 breast tumor cells compared to an isotypecontrol antibody (bottom) and this binding corresponded to cells thatexpress high levels of ESA and CD44 (top: CD44 x-axis and NOTCH1y-axis). (E) Anti-NOTCH1 antibody 31M80 showed increased binding todissociated colon tumor cells from two different patients compared to anisotype antibody control.

FIG. 3: Antibodies Against NOTCH1 EGF13 and EGF4 Fail to EffectivelyBlock Ligand Binding. NOTCH1 expressing HEK 293 cells were incubatedwith either DLL4-Fc (left) or JAG1-Fc (right) in the presence ofanti-NOTCH1 antibodies (13M57, 31M103, 31M106, or 31M108) or controlanti-DLL4 (21M18) or anti-JAG1 (64M14) antibodies. Binding of Fc fusionproteins to NOTCH1-expressing cells was detected by a PE-conjugated goatanti-Fc antibody and flow cytometry. Inhibition of ligand binding byanti-NOTCH1 antibodies was expressed as a percentage of inhibition bythe control ligand antibodies. Anti-NOTCH1 antibodies 31M103, 31M106,and 31M108, all of which specifically bind to EGF13, only partiallyinhibit ligand binding. 31M108 showed between 50-75% inhibition comparedto the ligand antibodies. Similarly, 31M103 and 31M106 showed onlybetween 25-50% inhibition. In contrast, 13M57 showed no inhibition ofligand binding when compared to inhibition by anti-DLL4 or anti-JAG1antibodies.

FIG. 4: Effect of NOTCH1 Monoclonal Antibodies against EGF4 on BreastTumor Cell Growth In Vitro. Breast tumor cells were cultured in thepresence of 2.5 ug/mL or 5 ug/mL anti-NOTCH1 antibody, control murineIgG or no antibody for three days followed by 18 hours of BrdU labeling.As shown on the top graphs, breast tumor cells cultured in the presenceof anti-NOTCH1 antibody 13M57 showed a decreased 450 nm/690 nmabsorbance ratio compared to controls. As a percentage of no antibodycontrol, the presence of anti-NOTCH1 antibodies resulted in astatistically significant decrease by one-way ANOVA followed by Tukey'stest in the proliferation of breast tumor cells compared to no antibodycontrol (p<0.5) or control IgG (p<0.05) (bottom).

FIG. 5: Effect of NOTCH1 Monoclonal Antibody 13M57 on PE-13 Tumor CellsIn Vivo. NOD/SCID mice injected with PE-13 tumor cells were treated withPBS or anti-NOTCH1 antibodies 3 days after cell injection, and thegrowth of tumor cells was determine twice a week. Total tumor volume wassignificantly reduced by 49% (p<0.05) in animals treated with anti-NOTCHantibodies 13M57 compared to PBS injected controls.

FIG. 6: Effect of NOTCH1 Monoclonal Antibodies on Colon Tumor Cells InVivo. NOD/SCID mice injected with OMP-C9 (A) or OMP-C8 (B) colon tumorcells were treated with PBS or anti-NOTCH1 13M57 antibodies (A) or13M57, 31M106, and 31M103 (B) three days after cell injection, and thegrowth of tumor cells was determine twice a week. Total tumor volume wassignificantly reduced in animals treated with anti-NOTCH antibodiescompared to PBS injected controls in both tumor models. Antibodiesagainst EGF4 and EGF13 were all equally effective against C8 colontumors (B).

FIG. 7: Effect of NOTCH1 Monoclonal Antibody 13M57 on PE-13 Tumor CellsExpressing Luciferase. NOD/SCID mice injected with PE-13 tumor cellswere treated with anti-NOTCH1 antibodies, control 5M108 controlantibodies, or PBS. A scale of luciferase activity is provided at theright of each picture with upper dark indicating the highest activity(100 or higher×10⁶) and lower levels (<30×10⁶) indicating low luciferasesignal. (A) Animals treated with PBS or 5M108 controls antibodies havetumors detected in the upper dark region of the scale. In contrast,tumors in animals treated with anti-Notch antibodies show luciferaseactivity mainly in the lower region of the scale. (B) Quantification ofluciferase signal shows total tumor volume was significantly reduced(p=0.04) in animals treated with anti-NOTCH1 antibodies 13M57 comparedto PBS and 5M108 injected controls.

FIG. 8: Effect of NOTCH2 Monoclonal Antibody 59M07 on Colon TumorGrowth. NOD/SCID mice injected with C6 colon tumor cells were treatedwith anti-NOTCH2 antibodies or PBS vehicle as a control. Animals treatedwith anti-NOTCH2 59M07 (triangles) showed significant reduction in tumorgrowth over 48 days compared to control treated animals (diamonds).

FIG. 9: Effect of anti-NOTCH1 Antibody 13M57 and ChemotherapyCombination Therapy on Breast Tumor Reoccurrence. (A) Graph of theresponse of UM-PE13 tumors to four different treatment regimes: Group 1:paclitaxel followed by PBS (squares); Group 2: paclitaxel followed by13M57 (inverted triangles); Group 3 paclitaxel+13M57 followed by PBS(circles); and Group 4: paclitaxel+13M57 followed by 13M57 (diamonds).Initial treatments commenced when the average tumor volume per group(n=10) was 130 mm³ (Arrow: Start Paclitaxel). Paclitaxel (orPaclitaxel+13M57) treatments were stopped at day 52 after the tumors hadregressed and were undetectable (Arrow: Stop Paclitaxel). (B) Both theindividual animal tumor volumes (dots) and average (lines) tumor volumesfor each treatment group are graphed. Concurrent combination treatmentfollowed by continual treatment with anti-NOTCH1 13M57 antibodies (farright) had the greatest effect on inhibiting tumor recurrence followingthe cessation of paclitaxel treatment.

FIG. 10: Effect of anti-NOTCH1 Antibody 13M57 and ChemotherapyCombination Therapy on Colon Tumor Growth. (A) A graph of C8 colon tumorvolume during and after twice weekly treatment with 7.5 mg/kgirinotecan, or with 10 mg/kg of the anti-NOTCH1 antibody 13M57 plus 7.5mg/kg irinotecan (n=10). The tumor volume of ten animals was assessedtwice per week. The last treatment dose was given on day 56 (*). (B) Agraph of C8 colon tumor volume during and after twice weekly treatmentwith 7.5 mg/kg irinotecan plus 10 mg/kg 13M57. The tumor volume of tenanimals was assessed twice per week. The last treatment dose was givenon day 56 (*). Concurrent combination treatment of irinotecan and 13M57prevented colon tumor growth and also maintained tumor cells in thisnon-proliferative state for up to thirty days post-treatment.

FIG. 11: Effect of anti-NOTCH1 Antibody 13M57 and ChemotherapyCombination Therapy on Established Colon Tumor Growth. A graph shows C8colon tumor volume over the course of treatment with either oxaliplatin(triangles), 10 mg/kg 13M57 (diamonds), a combination of oxaliplatin and13M57 (circles), or a control antibody (squares). Treatment with eitheranti-NOTCH1 13M57 or oxaliplatin significantly reduced tumor growth(p=0.04 vs. control), but combination treatment further reduced growthcompared to treatment with either agent alone (p=0.03 vs. single agent).

FIG. 12: Effect of NOTCH1 and NOTCH2 Antibody Combination Therapy onBreast Tumor Growth. (A) A graph of bioluminescence imaging of animalstreated either with 10 mg/kg anti-NOTCH1 31M108 (open triangles), 10mg/kg 59M07 anti-NOTCH2 (filled triangles), a combination of anti-NOTCH1and NOTCH2 antibodies (filled inverted triangles), or a control antibody(open circles). Animals were imaged twice weekly. Combination treatmentwith anti-NOTCH1 and NOTCH2 antibodies significantly reduced growth ofluciferase expressing PE13 tumor cells. (B) A graph of total tumorvolume of animals treated either with 10 mg/kg anti-NOTCH1 31M108 (opentriangles), 10 mg/kg 59M07 anti-NOTCH2 (filled triangles), a combinationof anti-NOTCH1 and NOTCH2 antibodies (filled inverted triangles), or acontrol antibody (open circles). Tumor volume was assessed twice perweek. Animals treated with 31M108 antibodies showed a significantreduction in total tumor volume compared to control treated animals(p<0.05). A further reduction of breast tumor growth was observed inanimals treated with a combination of anti-NOTCH1 and anti-NOTCH2antibodies as compared to treatment with either antibody alone (p<0.05).

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “antagonist” includes any molecule that partially or fullyblocks, inhibits, or neutralizes a biological activity of the Notchpathway. Suitable antagonist molecules specifically include antagonistantibodies or antibody fragments, fragments or amino acid sequencevariants of native Notch receptors. The term “antagonist” is used hereinto include any molecule that partially or fully blocks, inhibits, orneutralizes the expression of or the biological activity of a cancerstem cell marker disclosed herein and such biological activity includes,but is not limited to, inhibition of tumor growth.

The term “antibody” is used to mean an immunoglobulin molecule thatrecognizes and specifically binds to a target, such as a protein,polypeptide, peptide, carbohydrate, polynucleotide, lipid, orcombinations of the foregoing etc., through at least one antigenrecognition site within the variable region of the immunoglobulinmolecule. As used herein, the term encompasses intact polyclonalantibodies, intact monoclonal antibodies, antibody fragments (such asFab, Fab′, F(ab′)2, and Fv fragments), single chain Fv (scFv) mutants,multispecific antibodies such as bispecific antibodies generated from atleast two intact antibodies, fusion proteins comprising an antibodyportion, and any other modified immunoglobulin molecule comprising anantigen recognition site so long as the antibodies exhibit the desiredbiological activity. An antibody can be of any the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively. The different classes ofimmunoglobulins have different and well known subunit structures andthree-dimensional configurations. Antibodies can be naked or conjugatedto other molecules such as toxins, radioisotopes, etc.

As used herein, the term “antibody fragment” refers to a portion of anintact antibody and refers to the antigenic determining variable regionsof an intact antibody. Examples of antibody fragments include, but arenot limited to Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies,single chain antibodies, and multispecific antibodies formed fromantibody fragments.

An “Fv antibody” refers to the minimal antibody fragment that contains acomplete antigen-recognition and -binding site either as two-chains, inwhich one heavy and one light chain variable domain form a non-covalentdimer, or as a single-chain (scFv), in which one heavy and one lightchain variable domain are covalently linked by a flexible peptide linkerso that the two chains associate in a similar dimeric structure. In thisconfiguration the complementary determining regions (CDRs) of eachvariable domain interact to define the antigen-binding specificity ofthe Fv dimer. Alternatively a single variable domain (or half of an Fv)can be used to recognize and bind antigen, although generally with loweraffinity.

A “monoclonal antibody” as used herein refers to homogenous antibodypopulation involved in the highly specific recognition and binding of asingle antigenic determinant, or epitope. This is in contrast topolyclonal antibodies that typically include different antibodiesdirected against different antigenic determinants. The term “monoclonalantibody” encompasses both intact and full-length monoclonal antibodiesas well as antibody fragments (such as Fab, Fab′, F(ab′)2, Fv), singlechain (scFv) mutants, fusion proteins comprising an antibody portion,and any other modified immunoglobulin molecule comprising an antigenrecognition site. Furthermore, “monoclonal antibody” refers to suchantibodies made in any number of manners including but not limited to byhybridoma, phage selection, recombinant expression, and transgenicanimals.

As used herein, the term “humanized antibody” refers to forms ofnon-human (e.g. murine) antibodies that are specific immunoglobulinchains, chimeric immunoglobulins, or fragments thereof that containminimal non-human sequences. Typically, humanized antibodies are humanimmunoglobulins in which residues from the complementary determiningregion (CDR) are replaced by residues from the CDR of a non-humanspecies (e.g. mouse, rat, rabbit, hamster, etc.) that have the desiredspecificity, affinity, and capability. In some instances, the Fvframework region (FR) residues of a human immunoglobulin are replacedwith the corresponding residues in an antibody from a non-human speciesthat has the desired specificity, affinity, and capability. Thehumanized antibody can be further modified by the substitution ofadditional residue either in the Fv framework region and/or within thereplaced non-human residues to refine and optimize antibody specificity,affinity, and/or capability. In general, the humanized antibody willcomprise substantially all of at least one, and typically two or three,variable domains containing all or substantially all of the CDR regionsthat correspond to the non-human immunoglobulin whereas all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody can also comprise at least aportion of an immunoglobulin constant region or domain (Fc), typicallythat of a human immunoglobulin. Examples of methods used to generatehumanized antibodies are described in U.S. Pat. No. 5,225,539, hereinincorporated by reference.

The term “human antibody” as used herein means an antibody produced by ahuman or an antibody having an amino acid sequence corresponding to anantibody produced by a human made using any of the techniques known inthe art. This definition of a human antibody includes intact orfull-length antibodies, fragments thereof, and/or antibodies comprisingat least one human heavy and/or light chain polypeptide such as, forexample, an antibody comprising murine light chain and human heavy chainpolypeptides.

“Hybrid antibodies” are immunoglobulin molecules in which pairs of heavyand light chains from antibodies with different antigenic determinantregions are assembled together so that two different epitopes or twodifferent antigens can be recognized and bound by the resultingtetramer.

The term “chimeric antibodies” refers to antibodies wherein the aminoacid sequence of the immunoglobulin molecule is derived from two or morespecies. Typically, the variable region of both light and heavy chainscorresponds to the variable region of antibodies derived from onespecies of mammals (e.g. mouse, rat, rabbit, etc.) with the desiredspecificity, affinity, and capability while the constant regions arehomologous to the sequences in antibodies derived from another (usuallyhuman) to avoid eliciting an immune response in that species.

The term “epitope” or “antigenic determinant” are used interchangeablyherein and refer to that portion of an antigen capable of beingrecognized and specifically bound by a particular antibody. When theantigen is a polypeptide, epitopes can be formed both from contiguousamino acids and noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained upon protein denaturing, whereas epitopes formed by tertiaryfolding are typically lost upon protein denaturing. An epitope typicallyincludes at least 3, and more usually, at least 5 or 8-10 amino acids ina unique spatial conformation.

Competition between antibodies is determined by an assay in which theimmunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen. Numerous types of competitive bindingassays are known, for example: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al.,Methods in Enzymology 9:242-253 (1983)); solid phase directbiotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614-3619(1986)); solid phase direct labeled assay, solid phase direct labeledsandwich assay (see Harlow and Lane, “Antibodies, A Laboratory Manual,”Cold Spring Harbor Press (1988)); solid phase direct label RIA usingI-125 label (see Morel et al., Molec. Immunol. 25(1):7-15 (1988)); solidphase direct biotin-avidin EIA (Cheung et al., Virology 176:546-552(1990)); and direct labeled RIA (Moldenhauer et al., Scand. J. Immunol.32:77-82 (1990)). Typically, such an assay involves the use of purifiedantigen bound to a solid surface or cells bearing either of these, anunlabeled test immunoglobulin and a labeled reference immunoglobulin.Competitive inhibition is measured by determining the amount of labelbound to the solid surface or cells in the presence of the testimmunoglobulin. Usually the test immunoglobulin is present in excess.Antibodies identified by competition assay (competing antibodies)include antibodies binding to the same epitope as the reference antibodyand antibodies binding to an adjacent epitope sufficiently proximal tothe epitope bound by the reference antibody for steric hindrance tooccur. Usually, when a competing antibody is present in excess, it willinhibit specific binding of a reference antibody to a common antigen byat least 50 or 75%.

That an antibody “selectively binds” or “specifically binds” to anepitope or receptor means that the antibody reacts or associates morefrequently, more rapidly, with greater duration, with greater affinity,or with some combination of the above to the epitope or receptor thanwith alternative substances, including unrelated proteins. “Selectivelybinds” or “specifically binds” means, for instance, that an antibodybinds to a protein with a KD of at least about 0.1 mM, more usually atleast about 1 uM. “Selectively binds” or “specifically binds” means attimes that an antibody binds to a protein at times with a KD of at leastabout 0.1 uM or better, and at other times at least about 0.01 uM orbetter. Because of the sequence identity between homologous proteins indifferent species, specific binding can include an antibody thatrecognizes a cancer stem cell marker in more than one species.

As used herein, the terms “non-specific binding” and “backgroundbinding” when used in reference to the interaction of an antibody and aprotein or peptide refer to an interaction that is not dependent on thepresence of a particular structure (i.e., the antibody is binding toproteins in general rather that a particular structure such as anepitope).

The terms “isolated” or “purified” refer to material that issubstantially or essentially free from components that normallyaccompany it in its native state. Purity and homogeneity are typicallydetermined using analytical chemistry techniques such as polyacrylamidegel electrophoresis or high performance liquid chromatography. A protein(e.g. an antibody) or nucleic acid that is the predominant speciespresent in a preparation is substantially purified. In particular, anisolated nucleic acid is separated from open reading frames thatnaturally flank the gene and encode proteins other than protein encodedby the gene. An isolated antibody is separated from othernon-immunoglobulin proteins and from other immunoglobulin proteins withdifferent antigen binding specificity. It can also mean that the nucleicacid or protein is at least 85% pure, at least 95% pure, and in someembodiments, at least 99% pure.

As used herein, the terms “cancer” and “cancerous” refer to or describethe physiological condition in mammals in which a population of cellsare characterized by unregulated cell growth. Examples of cancerinclude, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia. More particular examples of such cancers include squamouscell cancer, small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma and various types of head and neckcancer.

The terms “proliferative disorder” and “proliferative disease” refer todisorders associated with abnormal cell proliferation such as cancer.

“Tumor” and “neoplasm” as used herein refer to any mass of tissue thatresult from excessive cell growth or proliferation, either benign(noncancerous) or malignant (cancerous) including pre-cancerous lesions.

“Metastasis” as used herein refers to the process by which a cancerspreads or transfers from the site of origin to other regions of thebody with the development of a similar cancerous lesion at the newlocation. A “metastatic” or “metastasizing” cell is one that losesadhesive contacts with neighboring cells and migrates via thebloodstream or lymph from the primary site of disease to invadeneighboring body structures.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

The terms “cancer stem cell”, “tumor stem cell”, or “solid tumor stemcell” are used interchangeably herein and refer to a population of cellsfrom a solid tumor that: (1) have extensive proliferative capacity; 2)are capable of asymmetric cell division to generate one or more kinds ofdifferentiated progeny with reduced proliferative or developmentalpotential; and (3) are capable of symmetric cell divisions forself-renewal or self-maintenance. These properties of “cancer stemcells”, “tumor stem cells” or “solid tumor stem cells” confer on thosecancer stem cells the ability to form palpable tumors upon serialtransplantation into an immunocompromised mouse compared to the majorityof tumor cells that fail to form tumors. Cancer stem cells undergoself-renewal versus differentiation in a chaotic manner to form tumorswith abnormal cell types that can change over time as mutations occur.The solid tumor stem cells of the present invention differ from the“cancer stem line” provided by U.S. Pat. No. 6,004,528. In that patent,the “cancer stem line” is defined as a slow growing progenitor cell typethat itself has few mutations but which undergoes symmetric rather thanasymmetric cell divisions as a result of tumorigenic changes that occurin the cell's environment. This “cancer stem line” hypothesis thusproposes that highly mutated, rapidly proliferating tumor cells ariselargely as a result of an abnormal environment, which causes relativelynormal stem cells to accumulate and then undergo mutations that causethem to become tumor cells. U.S. Pat. No. 6,004,528 proposes that such amodel can be used to enhance the diagnosis of cancer. The solid tumorstem cell model is fundamentally different than the “cancer stem line”model and as a result exhibits utilities not offered by the “cancer stemline” model. First, solid tumor stem cells are not “mutationallyspared”. The “mutationally spared cancer stem line” described by U.S.Pat. No. 6,004,528 can be considered a pre-cancerous lesion, while thesolid tumor stem cells described by this invention are cancer cells thatthemselves contain the mutations that are responsible for tumorigenesis.That is, the solid tumor stem cells (“cancer stem cells”) of theinvention would be included among the highly mutated cells that aredistinguished from the “cancer stem line” in U.S. Pat. No. 6,004,528.Second, the genetic mutations that lead to cancer can be largelyintrinsic within the solid tumor stem cells as well as beingenvironmental. The solid tumor stem cell model predicts that isolatedsolid tumor stem cells can give rise to additional tumors upontransplantation (thus explaining metastasis) while the “cancer stemline” model would predict that transplanted “cancer stem line” cellswould not be able to give rise to a new tumor, since it was theirabnormal environment that was tumorigenic. Indeed, the ability totransplant dissociated, and phenotypically isolated human solid tumorstem cells to mice (into an environment that is very different from thenormal tumor environment), where they still form new tumors,distinguishes the present invention from the “cancer stem line” model.Third, solid tumor stem cells likely divide both symmetrically andasymmetrically, such that symmetric cell division is not an obligateproperty. Fourth, solid tumor stem cells can divide rapidly or slowly,depending on many variables, such that a slow proliferation rate is nota defining characteristic.

The terms “cancer cell”, “tumor cell” and grammatical equivalents referto the total population of cells derived from a tumor including bothnon-tumorigenic cells, which comprise the bulk of the tumor cellpopulation, and tumorigenic stem cells (cancer stem cells).

As used herein “tumorigenic” refers to the functional features of asolid tumor stem cell including the properties of self-renewal (givingrise to additional tumorigenic cancer stem cells) and proliferation togenerate all other tumor cells (giving rise to differentiated and thusnon-tumorigenic tumor cells) that allow solid tumor stem cells to form atumor.

As used herein, the terms “stem cell cancer marker(s)”, “cancer stemcell marker(s)”, “tumor stem cell marker(s)”, or “solid tumor stem cellmarker(s)” refer to a gene or genes or a protein, polypeptide, orpeptide expressed by the gene or genes whose expression level, alone orin combination with other genes, is correlated with the presence oftumorigenic cancer cells compared to non-tumorigenic cells. Thecorrelation can relate to either an increased or decreased expression ofthe gene (e.g. increased or decreased levels of mRNA or the peptideencoded by the gene).

The terms “cancer stem cell gene signature”, “tumor stem cell genesignature” or “cancer stem cell signature” are used interchangeablyherein to refer to gene signatures comprising genes differentiallyexpressed in cancer stem cells compared to other cells or population ofcells, for example normal breast epithelial tissue. In some embodimentsthe cancer stem cell gene signatures comprise genes differentiallyexpressed in cancer stem cells versus normal breast epithelium by a foldchange, for example by 2 fold reduced and/or elevated expression, andfurther limited by using a statistical analysis such as, for example, bythe P value of a t-test across multiple samples. In another embodiment,the genes differentially expressed in cancer stem cells are divided intocancer stem cell gene signatures based on the correlation of theirexpression with a chosen gene in combination with their fold orpercentage expression change. Cancer stem cell signatures are predictiveboth retrospectively and prospectively of an aspect of clinicalvariability, including but not limited to metastasis and death.

The term “genetic test” as used herein refers to procedures whereby thegenetic make-up of a patient or a patient tumor sample is analyzed. Theanalysis can include detection of DNA, RNA, chromosomes, proteins ormetabolites to detect heritable or somatic disease-related genotypes orkaryotypes for clinical purposes.

As used herein, the terms “biopsy” or “biopsy tissue” refer to a sampleof tissue or fluid that is removed from a subject for the purpose ofdetermining if the sample contains cancerous tissue. In someembodiments, biopsy tissue or fluid is obtained because a subject issuspected of having cancer. The biopsy tissue or fluid is then examinedfor the presence or absence of cancer.

As used herein an “acceptable pharmaceutical carrier” refers to anymaterial that, when combined with an active ingredient of apharmaceutical composition such as an antibody, allows the antibody, forexample, to retain its biological activity. In addition, an “acceptablepharmaceutical carrier” does not trigger an immune response in arecipient subject. Examples include, but are not limited to, any of thestandard pharmaceutical carriers such as a phosphate buffered salinesolution, water, and various oil/water emulsions. Some diluents foraerosol or parenteral administration are phosphate buffered saline ornormal (0.9%) saline.

The term “therapeutically effective amount” refers to an amount of anantibody, polypeptide, polynucleotide, small organic molecule, or otherdrug effective to “treat” a disease or disorder in a subject or mammal.In the case of cancer, the therapeutically effective amount of the drugcan reduce the number of cancer cells; reduce the tumor size; inhibit orstop cancer cell infiltration into peripheral organs; inhibit and stoptumor metastasis; inhibit and stop tumor growth; relieve to some extentone or more of the symptoms associated with the cancer, or a combinationof such effects on cancer cells. To the extent the drug prevents growthand/or kills existing cancer cells, it can be referred to as cytostaticand/or cytotoxic.

As used herein, “providing a diagnosis” or “diagnostic information”refers to any information that is useful in determining whether apatient has a disease or condition and/or in classifying the disease orcondition into a phenotypic category or any category having significancewith regards to the prognosis of or likely response to treatment (eithertreatment in general or any particular treatment) of the disease orcondition. Similarly, diagnosis refers to providing any type ofdiagnostic information, including, but not limited to, whether a subjectis likely to have a condition (such as a tumor), information related tothe nature or classification of a tumor as for example a high risk tumoror a low risk tumor, information related to prognosis and/or informationuseful in selecting an appropriate treatment. Selection of treatment caninclude the choice of a particular chemotherapeutic agent or othertreatment modality such as surgery or radiation or a choice aboutwhether to withhold or deliver therapy.

As used herein, the terms “providing a prognosis”, “prognosticinformation”, or “predictive information” refer to providing informationregarding the impact of the presence of cancer (e.g., as determined bythe diagnostic methods of the present invention) on a subject's futurehealth (e.g., expected morbidity or mortality, the likelihood of gettingcancer, and the risk of metastasis).

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” refer to both 1) therapeutic measures that cure, slowdown, lessen symptoms of, and/or halt progression of a diagnosedpathologic condition or disorder and 2) prophylactic or preventativemeasures that prevent or slow the development of a targeted pathologiccondition or disorder. Thus those in need of treatment include thosealready with the disorder; those prone to have the disorder; and thosein whom the disorder is to be prevented. A subject is successfully“treated” according to the methods of the present invention if thepatient shows one or more of the following: a reduction in the number ofor complete absence of cancer cells; a reduction in the tumor size;inhibition of or an absence of cancer cell infiltration into peripheralorgans including the spread of cancer into soft tissue and bone;inhibition of or an absence of tumor metastasis; inhibition or anabsence of tumor growth; relief of one or more symptoms associated withthe specific cancer; reduced morbidity and mortality; and improvement inquality of life.

As used herein, the terms “polynucleotide” or “nucleic acid” refer to apolymer composed of a multiplicity of nucleotide units (ribonucleotideor deoxyribonucleotide or related structural variants) linked viaphosphodiester bonds, including but not limited to, DNA or RNA. The termencompasses sequences that include any of the known base analogs of DNAand RNA including, but not limited to, 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl 2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarbonylmethyluracil,5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyaceticacid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil,queosine, 2-thiocytosine, 5-methyl-2 thiouracil, 2-thiouracil,4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of apolypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction,immunogenicity, etc.) of the full-length or fragment are retained. Theterm also encompasses the coding region of a structural gene and thesequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb or more on either end such that thegene corresponds to the length of the full-length mRNA. Sequenceslocated 5′ of the coding region and present on the mRNA are referred toas 5′ non-translated sequences. Sequences located 3′ or downstream ofthe coding region and present on the mRNA are referred to as 3′non-translated sequences. The term “gene” encompasses both cDNA andgenomic forms of a gene. A genomic form or clone of a gene contains thecoding region interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns cancontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide. In addition to containing introns, genomic forms ofa gene can also include sequences located on both the 5′ and 3′ end ofthe sequences that are present on the RNA transcript. These sequencesare referred to as “flanking” sequences or regions (these flankingsequences are located 5′ or 3′ to the non-translated sequences presenton the mRNA transcript). The 5′ flanking region can contain regulatorysequences such as promoters and enhancers that control or influence thetranscription of the gene. The 3′ flanking region can contain sequencesthat direct the termination of transcription, post transcriptionalcleavage and polyadenylation.

The term “recombinant” when used with reference to a cell, nucleic acid,protein or vector indicates that the cell, nucleic acid, protein orvector has been modified by the introduction of a heterologous nucleicacid or protein, the alteration of a native nucleic acid or protein, orthat the cell is derived from a cell so modified. Thus, e.g.,recombinant cells express genes that are not found within the native(non-recombinant) form of the cell or express native genes that areoverexpressed or otherwise abnormally expressed such as, for example,expressed as non-naturally occurring fragments or splice variants. Bythe term “recombinant nucleic acid” herein is meant nucleic acid,originally formed in vitro, in general, by the manipulation of nucleicacid, e.g., using polymerases and endonucleases, in a form not normallyfound in nature. In this manner, operably linkage of different sequencesis achieved. Thus an isolated nucleic acid, in a linear form, or anexpression vector formed in vitro by ligating DNA molecules that are notnormally joined, are both considered recombinant for the purposes ofthis invention. It is understood that once a recombinant nucleic acid ismade and introduced into a host cell or organism, it will replicatenon-recombinantly, i.e., using the in vivo cellular machinery of thehost cell rather than in vitro manipulations; however, such nucleicacids, once produced recombinantly, although subsequently replicatednon-recombinantly, are still considered recombinant for the purposes ofthe invention. Similarly, a “recombinant protein” is a protein madeusing recombinant techniques, i.e., through the expression of arecombinant nucleic acid as depicted above.

As used herein, the term “heterologous gene” refers to a gene that isnot in its natural environment. For example, a heterologous geneincludes a gene from one species introduced into another species. Aheterologous gene also includes a gene native to an organism that hasbeen altered in some way (e.g., mutated, added in multiple copies,linked to non-native regulatory sequences, etc). Heterologous genes aredistinguished from endogenous genes in that the heterologous genesequences are typically joined to DNA sequences that are not foundnaturally associated with the gene sequences in the chromosome or areassociated with portions of the chromosome not found in nature (e.g.,genes expressed in loci where the gene is not normally expressed).

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vehicle” is sometimes used interchangeably with “vector.” Vectorsare often derived from plasmids, bacteriophages, or plant or animalviruses.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments. Unless otherwise provided,ligation can be accomplished using known buffers and conditions with 10units to T4 DNA ligase (“ligase”) per 0.5 ug of approximately equimolaramounts of the DNA fragments to be ligated. Ligation of nucleic acid canserve to link two proteins together in-frame to produce a singleprotein, or fusion protein.

As used herein, the term “gene expression” refers to the process ofconverting genetic information encoded in a gene into RNA (e.g., mRNA,rRNA, tRNA, or snRNA) through “transcription” of the gene (e.g., via theenzymatic action of an RNA polymerase), and for protein encoding genes,into protein through “translation” of mRNA. Gene expression can beregulated at many stages in the process. “Up-regulation” or “activation”refers to regulation that increases the production of gene expressionproducts (e.g., RNA or protein), while “down-regulation” or “repression”refers to regulation that decrease production. Molecules (e.g.,transcription factors) that are involved in upregulation ordown-regulation are often called “activators” and “repressors,”respectively.

The terms “polypeptide,” “peptide,” “protein”, and “protein fragment”are used interchangeably herein to refer to a polymer of amino acidresidues. The terms apply to amino acid polymers in which one or moreamino acid residue is an artificial chemical mimetic of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers and non-naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs refersto compounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an alpha carbon that is bound to a hydrogen,a carboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs can have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. Amino acid mimetics refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions similarly to anaturally occurring amino acid.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. “Amino acid variants” refers to amino acidsequences. With respect to particular nucleic acid sequences,conservatively modified variants refers to those nucleic acids whichencode identical or essentially identical amino acid sequences, or wherethe nucleic acid does not encode an amino acid sequence, to essentiallyidentical or associated (e.g., naturally contiguous) sequences. Becauseof the degeneracy of the genetic code, a large number of functionallyidentical nucleic acids encode most proteins. For instance, the codonsGCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at everyposition where an alanine is specified by a codon, the codon can bealtered to another of the corresponding codons described withoutaltering the encoded polypeptide. Such nucleic acid variations are“silent variations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein which encodes apolypeptide also describes silent variations of the nucleic acid. It isrecognized that in certain contexts each codon in a nucleic acid (exceptAUG, which is ordinarily the only codon for methionine, and TGG, whichis ordinarily the only codon for tryptophan) can be modified to yield afunctionally identical molecule. Accordingly, silent variations of anucleic acid which encodes a polypeptide is implicit in a describedsequence with respect to the expression product, but not with respect toactual probe sequences. As to amino acid sequences, it will berecognized that individual substitutions, deletions or additions to anucleic acid, peptide, polypeptide, or protein sequence which alters,adds or deletes a single amino acid or a small percentage of amino acidsin the encoded sequence is a “conservatively modified variant” includingwhere the alteration results in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention. Typically conservative substitutions include: 1) Alanine (A),Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (19841).

The term “epitope tagged” as used herein refers to a chimericpolypeptide comprising a cancer stem cell marker protein, or a domainsequence or portion thereof, fused to an “epitope tag”. The epitope tagpolypeptide comprises enough amino acid residues to provide an epitopefor recognition by an antibody, yet is short enough such that it doesnot interfere with the activity of the cancer stem cell marker protein.Suitable epitope tags generally have at least six amino acid residues,usually between about 8 to about 50 amino acid residues, and at timesbetween about 10 to about 20 residues. Commonly used epitope tagsinclude Fc, HA, H is, and FLAG tags.

Certain Embodiments of the Present Invention

The present invention provides compositions and methods for studying,diagnosing, characterizing, and treating cancer. In particular, thepresent invention provides antagonists against solid tumor stem cellmarkers and methods of using these antagonists to inhibit tumor growthand treat cancer in human patients. Several antagonists includeantibodies that specifically recognize solid tumor stem cell markerproteins.

The present invention further identifies molecules (e.g. antibodies)that specifically bind to a non-ligand binding region of theextracellular domain of a human NOTCH receptor and inhibit tumor growthin vivo. The ligand binding region of Notch, which is necessary andsufficient for ligand binding, has been identified as EGF repeats 11 and12, suggesting this region of the Notch receptor is important in Notchsignaling and turmorigenesis (Rebay et al., 1991, Cell 67:687; Lei etal., 2003, Dev. 130:6411; Hambleton et al., 2004, Structure 12:2173).Unexpectedly and for the first time, antibodies that bind outside theligand binding domain of the extracellular domain of human Notchreceptor were found to inhibit tumor cell growth in vivo. One suchantibody to an epitope within EGF repeat 4 of NOTCH1 inhibited tumorcell growth in an animal model. These results suggest that antibodiesthat bind outside the ligand binding domain of the extracellular domainof one or more of the human Notch receptors—NOTCH1, NOTCH2, NOTCH3, andNOTCH4—have value as potential cancer therapeutics.

In certain embodiments, the present invention provides an antibody thatspecifically binds to a non-ligand binding region of the extracellulardomain of a human NOTCH receptor and inhibits growth of tumor cells. Incertain embodiments, the antibody binds to a non-ligand binding regionof the extracellular domain of NOTCH1 receptor. In certain embodiments,the antibody that specifically binds to a non-ligand binding region ofthe extracellular domain of a human NOTCH receptor and inhibits growthof tumor cells specifically binds to a non-ligand binding region of theextracellular domain of at least two Notch receptor family members.

In certain embodiments, the antibody that specifically binds to anon-ligand binding region of the extracellular domain of a human NOTCHreceptor and inhibits growth of tumor cells is a monoclonal antibody. Incertain embodiments, the antibody that specifically binds to anon-ligand binding region of the extracellular domain of a human NOTCHreceptor and inhibits growth of tumor cells is a chimeric antibody. Incertain embodiments, the antibody that specifically binds to anon-ligand binding region of the extracellular domain of a human NOTCHreceptor and inhibits growth of tumor cells is a humanized antibody. Incertain embodiments, the antibody that specifically binds to anon-ligand binding region of the extracellular domain of a human NOTCHreceptor and inhibits growth of tumor cells is a human antibody. Incertain embodiments, the present invention provides a hybridomaproducing an antibody that specifically binds to a non-ligand bindingregion of the extracellular domain of a human NOTCH receptor andinhibits growth of tumor cells.

In certain embodiments the present invention provides an antibody thatspecifically binds to a non-ligand binding region comprising EGF repeats1-10 of the extracellular domain of a human NOTCH receptor and inhibitsgrowth of tumor cells. In certain embodiments the present inventionprovides an antibody that specifically binds to a non-ligand bindingregion comprising EGF repeats 13-36 of the extracellular domain of ahuman NOTCH receptor and inhibits growth of tumor cells. Certainembodiments provide an antibody that specifically binds to a non-ligandbinding region comprising EGF repeats 4 of the extracellular domain of ahuman NOTCH receptor and inhibits growth of tumor cells. Certainembodiments provide an antibody that specifically binds to a non-ligandbinding region comprising EGF repeats 13 of the extracellular domain ofa human NOTCH receptor and inhibits growth of tumor cells.

In certain embodiments, the present invention provides an isolatedpolypeptide that specifically binds to a non-ligand binding region of anextracellular domain of a human NOTCH receptor comprising: (a) a heavychain variable region having CDR sequences set forth in SEQ ID NOS: 12,13, and 14; and (b) a light chain variable region having CDR sequencesset forth in SEQ ID NOS: 15, 16, and 17. In certain embodiments, theisolated polypeptide that specifically binds to a non-ligand bindingregion of an extracellular domain of a human NOTCH receptor comprises:(a) heavy chains set forth in SEQ ID NOS: 4 and 5; and (b) light chainsset forth in SEQ ID NOS: 6 and 7.

In certain embodiments the present invention provides a method oftreating cancer to a subject in need thereof comprising administering atherapeutically effective amount of an antibody that specifically bindsto a non-ligand binding region of the extracellular domain of a humanNOTCH receptor protein to the subject and inhibits growth of tumor cellsin the subject. In certain embodiments, the method of treating cancer ina subject in need thereof comprises administering a therapeuticallyeffective amount of an antibody to the subject that specifically bindsto a non-ligand binding region of the extracellular domain of NOTCH1receptor and inhibits growth of tumor cells. In certain embodiments, themethod of treating cancer comprises administering a therapeuticallyeffective amount of an antibody that specifically binds to at least twoNotch receptor family members and inhibits growth of tumor cells.

In certain embodiments, the method of treating cancer comprisesadministering a therapeutically effective amount of a monoclonalantibody that specifically binds to a non-ligand binding region of theextracellular domain of a human NOTCH receptor and inhibits growth oftumor cells. In certain embodiments, the method of treating cancercomprises administering a therapeutically effective amount of a chimericantibody that specifically binds to a non-ligand binding region of theextracellular domain of a human NOTCH receptor and inhibits growth oftumor cells. In certain embodiments, the method of treating cancercomprises administering a therapeutically effective amount of ahumanized antibody that specifically binds to a non-ligand bindingregion of the extracellular domain of a human NOTCH receptor andinhibits growth of tumor cells. In certain embodiments, the method oftreating cancer comprises administering a therapeutically effectiveamount of a human antibody that specifically binds to a non-ligandbinding region of the extracellular domain of a human NOTCH receptor andinhibits growth of tumor cells.

In certain embodiments, the method of treating cancer comprisesadministering a therapeutically effective amount of an antibody thatspecifically binds to a non-ligand binding region of the extracellulardomain of a human NOTCH receptor comprising EGF repeats 1-10 andinhibits growth of tumor cells. In certain embodiments, the method oftreating cancer comprises administering a therapeutically effectiveamount of an antibody that specifically binds to a non-ligand bindingregion of the extracellular domain of a human NOTCH receptor comprisingEGF repeats 13-36 and inhibits growth of tumor cells. In certainembodiments, the method of treating cancer comprises administering atherapeutically effective amount of an antibody that specifically bindsto a non-ligand binding region of the extracellular domain of a humanNOTCH receptor comprising EGF repeats 4 and inhibits growth of tumorcells. In certain embodiments, the method of treating cancer comprisesadministering a therapeutically effective amount of an antibody thatspecifically binds to a non-ligand binding region of the extracellulardomain of a human NOTCH receptor comprising EGF repeats 4 and inhibitsgrowth of tumor cells.

In certain embodiments, the method of treating cancer comprisesadministering a therapeutically effective amount of an isolatedpolypeptide that specifically binds to a non-ligand binding region ofthe extracellular domain of a human NOTCH receptor comprising: (a) aheavy chain variable region having CDR sequences set forth in SEQ IDNOS: 12, 13, and 14; and (b) a light chain variable region having CDRsequences set forth in SEQ ID NOS: 15, 16, and 17 and inhibits growth oftumor cells. In certain embodiments, the method of treating cancercomprises administering a therapeutically effective amount of anantibody that specifically binds to a non-ligand binding region of theextracellular domain of a human NOTCH receptor comprising (a) heavychains set forth in SEQ ID NOS: 4 and 5; and (b) light chains set forthin SEQ ID NOS: 6 and 7.

In certain embodiments, the method of treating cancer comprisesadministering a therapeutically effective amount of an antibodyconjugated to a cytotoxic moiety that specifically binds to a non-ligandbinding region of the extracellular domain of a human NOTCH receptor andinhibits growth of tumor cells. In certain embodiments, the method oftreating cancer comprises administering a therapeutically effectiveamount of an antibody that specifically binds to a non-ligand bindingregion of the extracellular domain of a human NOTCH receptor andinhibits growth of tumor cells in combination with radiation therapy. Incertain embodiments, the method of treating cancer comprisesadministering a therapeutically effective amount of an antibody thatspecifically binds to a non-ligand binding region of the extracellulardomain of a human NOTCH receptor and inhibits growth of tumor cells incombination with chemotherapy. In certain embodiments, the method oftreating cancer comprises administering a therapeutically effectiveamount of an antibody that specifically binds to a non-ligand bindingregion of the extracellular domain of a human NOTCH receptor andinhibits growth of tumor cells that are from a breast tumor, colorectaltumor, lung tumor, pancreatic tumor, prostate tumor, or a head and necktumor.

In certain embodiments, the method of treating cancer comprisesidentifying patients using a genetic test for treatment with theantibody that specifically binds to a non-ligand binding region of theextracellular domain of a human NOTCH receptor; and administering atherapeutically effective amount of an antibody that specifically bindsto a non-ligand binding region of the extracellular domain of a humanNOTCH receptor and inhibits growth of tumor cells. In certainembodiments, the method of treating cancer comprises identify patientsfor treatment with the antibody that specifically binds to a non-ligandbinding region of the extracellular domain of a human NOTCH receptorusing a genetic test that detects a cancer stem cell signature; andadministering a therapeutically effective amount of an antibody thatspecifically binds to a non-ligand binding region of the extracellulardomain of a human NOTCH receptor and inhibits growth of tumor cells.

In certain embodiments, the present invention provides a method ofidentifying a molecule that binds to a non-ligand binding region of anextracellular domain of a human NOTCH receptor and inhibits growth oftumor cells, the method comprising: i) incubating the molecule with thenon-ligand binding domain of the extracellular domain of a human Notchreceptor; ii) determining if the molecule binds to the non-ligandbinding region of the extracellular domain of the human Notch receptor;and iii) determining if the molecule inhibits growth of tumor cells. Incertain embodiments, the invention provides a method of identifying amolecule that binds to a non-ligand binding region of an extracellulardomain of a human NOTCH receptor and inhibits growth of tumor cells, themethod comprising: i) incubating the molecule with the non-ligandbinding domain of the extracellular domain of a human Notch receptorcomprising EGF repeats 1-10; ii) determining if the molecule binds tothe non-ligand binding region of the extracellular domain of the humanNotch receptor comprising EGF repeats 1-10; and iii) determining if themolecule inhibits growth of tumor cells. In certain embodiments, theinvention provides a method of identifying a molecule that binds to anon-ligand binding region of an extracellular domain of a human NOTCHreceptor and inhibits growth of tumor cells, the method comprising: i)incubating the molecule with the non-ligand binding domain of theextracellular domain of a human Notch receptor comprising EGF repeats13-36; ii) determining if the molecule binds to the non-ligand bindingregion of the extracellular domain of the human Notch receptorcomprising EGF repeats 13-36; and iii) determining if the moleculeinhibits growth of tumor cells.

In certain embodiments, the present invention provides a pharmaceuticalcomposition comprising an antibody that specifically binds to anon-ligand binding region of the extracellular domain of a human NOTCHreceptor and inhibits growth of tumor cells.

In certain embodiments, the present invention provides a method ofmaking an antibody that specifically binds to a non-ligand bindingregion of the extracellular domain of a human NOTCH receptor andinhibits growth of tumor cells.

In certain embodiments, the present invention provides an isolatednucleic acid that encodes an antibody that specifically binds to anon-ligand binding region of the extracellular domain of a human NOTCHreceptor and inhibits growth of tumor cells.

Stem Cells and Solid Tumor Stem Cells

Common cancers arise in tissues that contain a subpopulation ofproliferating cells that are responsible for replenishing theshort-lived mature cells. In such organs, cell maturation is arranged ina hierarchy in which a rare population of stem cells give rise both tothe more differentiated cells and perpetuate themselves through aprocess called self renewal (Akashi & Weissman, Developmental Biology ofHematopoiesis, Oxford Univ. Press, NY, 2001; Spangrude et al., 1988,Science 241:58-61; Baum et al., 1992, PNAS 89:2804-8; Morrison et al.,1995, PNAS 92:10302-6; Morrison et al., 1996, Immunity 5:207-16;Morrison et al., 1995, Annu. Rev. Cell Dev. Biol. 11:35-71; Morrison etal., 1997, Dev. 124:1929-39; Morrison & Weissman, 1994, Immunity 1:661;Morrison et al., 1997, Cell 88:287-98; Uchida et al., 2000, PNAS97:14720-5; Morrison et al., 2000, Cell 101:499-510). Although it islikely that most tissues contain stem cells, due to their rarity thesecells have been rigorously identified and purified to study theirbiological, molecular, and biochemical properties in only a few tissues.The best characterized stem cells are those that give rise to thehematopoietic system, called hematopoietic stem cells (HSCs). Theutility of HSCs has been demonstrated in cancer therapy with theirextensive use for bone marrow transplantation to regenerate thehematolymphoid system following myeloablative protocols (Baum et al.,Bone Marrow Transplantation, Blackwell Scientific Publications, Boston,1994). Understanding the cellular biology of the tissues in whichcancers arise, and specifically of the stem cells residing in thosetissues, promises to provide new insights into cancer biology.

Like the tissues in which they originate, solid tumors consist of aheterogeneous population of cells. That the majority of these cells lacktumorigenicity suggested that the development and maintenance of solidtumors also relies on a small population of stem cells (i.e.,tumorigenic cancer cells) with the capacity to proliferate andefficiently give rise both to additional tumor stem cells (self-renewal)and to the majority of more differentiated tumor cells that lacktumorigenic potential (i.e., non-tumorigenic cancer cells). The conceptof cancer stem cells was first introduced soon after the discovery ofHSC and was established experimentally in acute myelogenous leukemia(AML) (Park et al., 1971, J. Natl. Cancer Inst. 46:411-22; Lapidot etal., 1994, Nature 367:645-8; Bonnet & Dick, 1997, Nat. Med. 3:730-7;Hope et al., 2004, Nat. Immunol. 5:738-43). Stem cells from solid tumorshave more recently been isolated based on their expression of a uniquepattern of cell-surface receptors and on the assessment of theirproperties of self-renewal and proliferation in culture and in xenograftanimal models. An ESA+ CD44+ CD24−/low Lineage-population greater than50-fold enriched for the ability to form tumors relative tounfractionated tumor cells was discovered (Al-Hajj et al., 2003, PNAS100:3983-8). The ability to isolate tumorigenic cancer stem cells fromthe bulk of non-tumorigenic tumor cells has led to the identification ofcancer stem cell markers, genes with differential expression in cancerstem cells compared to non-tumorigenic tumor cells or normal breastepithelium, using microarray analysis. The present invention employs theknowledge of these identified cancer stem cell markers to study,characterize, diagnosis and treat cancer.

Cancer Stem Cell Marker Protein

Normal stem cells and cancer stem cells share the ability to proliferateand self-renew, thus it is not surprising that a number of genes thatregulate normal stem cell development contribute to tumorigenesis(reviewed in Reya et al., 2001, Nature 414:105-111 and Taipale & Beachy,2001, Nature 411:349-354). The present invention identifies Notchreceptor, for example, Notch1 as a marker of cancer stem cells,implicating the Notch signaling pathway in the maintenance of cancerstem cells and as a target for treating cancer via the elimination ofthese tumorigenic cells.

The Notch signaling pathway is one of several critical regulators ofembryonic pattern formation, post-embryonic tissue maintenance, and stemcell biology. More specifically, Notch signaling is involved in theprocess of lateral inhibition between adjacent cell fates and plays animportant role in cell fate determination during asymmetric celldivisions. Unregulated Notch signaling is associated with numerous humancancers where it can alter the developmental fate of tumor cells tomaintain them in an undifferentiated and proliferative state (Brennanand Brown, 2003, Breast Cancer Res. 5:69). Thus carcinogenesis canproceed by usurping homeostatic mechanisms controlling normaldevelopment and tissue repair by stem cell populations (Beachy et al.,2004, Nature 432:324).

The Notch receptor was first identified in Drosophila mutants withhaploinsufficiency resulting in notches at the wing margin whereasloss-of-function producing an embryonic lethal “neurogenic” phenotypewhere cells of the epidermis switch fate to neural tissue (Moohr, 1919,Genet. 4:252; Poulson, 1937, PNAS 23:133; Poulson, 1940, J. Exp. Zool.83:271). The Notch receptor is a single-pass transmembrane receptorcontaining numerous tandem epidermal growth factor (EGF)-like repeatsand cysteine-rich Notch/LIN-12 repeats within a large extracellulardomain (Wharton et al., 1985, Cell 43:567; Kidd et al., 1986, Mol. Cell.Biol. 6:3094; reviewed in Artavanis et al., 1999, Science 284:770). Fourmammalian Notch proteins have been identified (NOTCH1, NOTCH2, NOTCH3,and NOTCH4), and mutations in these receptors invariably result indevelopmental abnormalities and human pathologies including severalcancers as described in detail below (Gridley, 1997, Mol. Cell.Neurosci. 9:103; Joutel & Tournier-Lasserve, 1998, Semin. Cell Dev.Biol. 9:619-25).

The Notch receptor is activated by single-pass transmembrane ligands ofthe Delta, Serrated, Lag-2 (DSL) family. There are five known Notchligands in mammals: Delta-like 1 (Dll1), Delta-like 3 (Dll3), Delta-like4 (Dll4), Jagged 1 and Jagged 2 characterized by a DSL domain and tandemEGF-like repeats within the extracellular domain. The extracellulardomain of the Notch receptor interacts with that of its ligands,typically on adjacent cells, resulting in two proteolytic cleavages ofNotch, one extracellular mediated by an ADAM protease and one within thetransmembrane domain mediated by gamma secretase. This latter cleavagegenerates the Notch intracellular domain (NICD), which then enters thenucleus where it activates the CBF1, Suppressor of Hairless [Su(H)],Lag-2 (CSL) family of transcription factors as the major downstreameffectors to increase transcription of nuclear basic helix-loop-helixtranscription factors of the Hairy and Enhancer of Split [E(spl)] family(Artavanis et al., 1999, Science 284:770; Brennan and Brown, 2003,Breast Cancer Res. 5:69; Iso et al., 2003, Arterioscler. Thromb. Vasc.Biol. 23:543). Alternative intracellular pathways involving thecytoplasmic protein Deltex identified in Drosophila may also exist inmammals (Martinez et al., 2002, Curr. Opin. Genet. Dev. 12:524-33), andthis Deltex-dependent pathway may act to suppress expression of Wnttarget genes (Brennan et al., 1999, Curr. Biol. 9:707-710; Lawrence etal., 2001, Curr. Biol. 11:375-85).

Hematopoietic stem cells (HSCs) are the best understood stem cells inthe body, and Notch signaling is implicated both in their normalmaintenance as well as in leukemic transformation (Kopper & Hajdu, 2004,Pathol. Oncol. Res. 10:69-73). HSCs are a rare population of cells thatreside in a stromal niche within the adult bone marrow. These cells arecharacterized both by a unique gene expression profile as well as anability to continuously give rise to more differentiated progenitorcells to reconstitute the entire hematopoietic system. Constitutiveactivation of Notch1 signaling in HSCs and progenitor cells establishesimmortalized cell lines that generate both lymphoid and myeloid cells invitro and in long-term reconstitution assays (Varnum-Finney et al.,2000, Nat. Med. 6:1278-81), and the presence of Jagged 1 increasesengraftment of human bone marrow cell populations enriched for HSCs(Karanu et al., 2000, J. Exp. Med. 192:1365-72). More recently, Notchsignaling has been demonstrate in HSCs in vivo and shown to be involvedin inhibiting HSC differentiation. Furthermore, Notch signaling appearsto be required for Wnt-mediated HSC self-renewal (Duncan et al., 2005,Nat. Immunol. 6:314).

The Notch signaling pathway also plays a central role in the maintenanceof neural stem cells is implicated both in their normal maintenance aswell as in brain cancers (Kopper & Hajdu, 2004, Pathol. Oncol. Res.10:69-73; Purow et al., 2005, Cancer Res. 65:2353-63; Hallahan et al.,2004, Cancer Res. 64:7794-800). Neural stem cells give rise to allneuronal and glial cells in the mammalian nervous system duringdevelopment, and more recently have been identified in the adult brain(Gage, 2000, Science 287:1433-8). Mice deficient for Notch1; the Notchtarget genes Hes1, 3, and 5; and a regulator of Notch signalingpresenilin1 (PS1) show decreased numbers of embryonic neural stem cells.Furthermore, adult neural stem cells are reduced in the brains of PS1heterozygote mice (Nakamura et al., 2000, J. Neurosci. 20:283-93;Hitoshi et al., 2002, Genes Dev. 16:846-58). The reduction in neuralstem cells appears to result from their premature differentiation intoneurons (Hatakeyama et al., 2004, Dev. 131:5539-50) suggesting thatNotch signaling regulates neural stem cell differentiation andself-renewal.

Aberrant Notch signaling is implicated in a number of human cancers. TheNOTCH1 gene in humans was first identified in a subset of T-cell acutelymphoblastic leukemias as a translocated locus resulting in activationof the Notch pathway (Ellisen et al., 1991, Cell 66:649-61).Constitutive activation of Notch1 signaling in T-cells in mouse modelssimilarly generates T-cell lymphomas suggesting a causative role (Robeyet al., 1996, Cell 87:483-92; Pear et al., 1996, J. Exp. Med.183:2283-91; Yan et al., 2001, Blood 98:3793-9; Bellavia et al., 2000,EMBO J. 19:3337-48). Recently NOTCH1 point mutations, insertions, anddeletions producing aberrant NOTCH1 signaling have been found to befrequently present in both childhood and adult T-cell acutelymphoblastic leukemia/lymphoma (Pear & Aster, 2004, Curr. Opin.Hematol. 11:416-33).

The frequent insertion of the mouse mammary tumor virus into both theNotch1 and Notch4 locus in mammary tumors and the resulting activatedNotch protein fragments first implicated Notch signaling in breastcancer (Gallahan & Callahan, 1987, J. Virol. 61:66-74; Brennan & Brown,2003, Breast Cancer Res. 5:69; Politi et al., 2004, Semin. Cancer Biol.14:341-7). Further studies in transgenic mice have confirmed a role forNotch in ductal branching during normal mammary gland development, and aconstitutively active form of Notch4 in mammary epithelial cellsinhibits epithelial differentiation and results in tumorigenesis(Jhappan et al., 1992, Genes & Dev. 6:345-5; Gallahan et al., 1996,Cancer Res. 56:1775-85; Smith et al., 1995, Cell Growth Differ.6:563-77; Soriano et al., 2000, Int. J. Cancer 86:652-9; Uyttendaele etal., 1998, Dev. Biol. 196:204-17; Politi et al., 2004, Semin. CancerBiol. 14:341-7). Currently the evidence for a role for Notch in humanbreast cancer is limited to the expression of Notch receptors in breastcarcinomas and their correlation with clinical outcome (Weijzen et al.,2002, Nat. Med. 8:979-86; Parr et al., 2004, Int. J. Mol. Med.14:779-86). Furthermore, overexpression of the Notch pathway has beenobserved in cervical cancers (Zagouras et al., 1995, PNAS 92:6414-8),renal cell carcinomas (Rae et al., 2000, Int. J. Cancer 88:726-32), headand neck squamous cell carcinomas (Leethanakul et al., 2000, Oncogene19:3220-4), endometrial cancers (Suzuki et al., 2000, Int. J. Oncol.17:1131-9), and neuroblastomas (van Limpt et al., 2000, Med. Pediatr.Oncol. 35:554-8) suggestive of a potential role for Notch in thedevelopment of a number of neoplasms. Interestingly, Notch signalingmight play a role in the maintenance of the undifferentiated state ofApc-mutant neoplastic cells of the colon (van Es & Clevers, 2005, Trendsin Mol. Med. 11:496-502).

The Notch pathway is also involved in multiple aspects of vasculardevelopment including proliferation, migration, smooth muscledifferentiation, angiogenesis and arterial-venous differentiation (Isoet al., 2003, Arterioscler. Thromb. Vasc. Biol. 23:543). For example,homozygous null mutations in Notch-1/4 and Jagged-1 as well asheterozygous loss of Dll4 result in severe though variable defects inarterial development and yolk sac vascularization. Furthermore,Dll1-deficient and Notch-2-hypomorphic mice embryos show hemorrhage thatlikely results from poor development of vascular structures (Gale etal., 2004, PNAS, 101:15949-54; Krebs et al., 2000, Genes Dev.14:1343-52; Xue et al., 1999, Hum. Mel Genet. 8:723-30; Hrabe de Angeliset al., 1997, Nature 386:717-21; McCright et al., 2001, Dev.128:491-502). In human, mutations in JAGGED1 are associated withAlagille syndrome, a developmental disorder that includes vasculardefects, and mutations in NOTCH3 are responsible for an inheritedvascular dementia (CADASIL) in which vessel homeostasis is defective(Joutel et al., 1996, Nature 383:707-10).

The identification of NOTCH1, NOTCH4, DLL1 and DLL4 as genes expressedin cancer stem cells compared to normal breast epithelium suggests thattargeting the Notch pathway can help eliminate not only the majority ofnontumorigenic cancer cells, but the tumorigenic cells responsible forthe formation and reoccurrence of solid tumors. Furthermore, because ofthe prominent role of angiogenesis in tumor formation and maintenance,targeting the Notch pathway can also effectively inhibit angiogenesis,starving a cancer of nutrients and contributing to its elimination.

Diagnostic Assays

The present invention provides a cancer stem cell marker the expressionof which can be analyzed to detect, characterize, diagnosis or monitor adisease associated with expression of a cancer stem cell marker. In someembodiments, expression of a cancer stem cell marker is determined bypolynucleotide expression such as, for example, mRNA encoding the cancerstem cell marker. The polynucleotide can be detected and quantified byany of a number of means well known in the art. In some embodiments,mRNA encoding a cancer stem cell marker is detected by in situhybridization of tissue sections from, from example, a patient biopsy.Alternatively, RNA can be isolated from a tissue and detected by, forexample, Northern blot, quantitative RT-PCR or microarrays. For example,total RNA can be extracted from a tissue sample and primers thatspecifically hybridize and amplify a cancer stem cell marker can be usedto detect expression of a cancer stem cell marker polynucleotide usingRT-PCR.

In other embodiments, expression of a cancer stem cell marker can bedetermined by detection of the corresponding polypeptide. Thepolypeptide can be detected and quantified by any of a number of meanswell known in the art. In some embodiments, a cancer stem cell markerpolypeptide is detected using analytic biochemical methods such as, forexample, electrophoresis, capillary electrophoresis, high performanceliquid chromatography (HPLC) or thin layer chromatography (TLC). Theisolated polypeptide can also be sequenced according to standardtechniques. In some embodiments, a cancer stem cell marker protein isdetected with antibodies raised against the protein using, for example,immunofluorescence or immunohistochemistry on tissue sections.Alternatively antibodies against a cancer stem cell marker can detectexpression using, for example, ELISA, FACS, Western blot,immunoprecipitation or protein microarrays. For example, cancer stemcells can be isolated from a patient biopsy and expression of a cancerstem cell marker protein detected with fluorescently labeled antibodiesusing FACS. In another method, the cells expressing a cancer stem cellmarker can be detected in vivo using labeled antibodies in typicalimaging system. For example, antibodies labeled with paramagneticisotopes can be used for magnetic resonance imaging (MRI).

In some embodiments of the present invention, a diagnostic assaycomprises determining the expression or not of a cancer stem cell markerin tumor cells using, for example, immunohistochemistry, in situhybridization, or RT-PCR. In other embodiments, a diagnostic assaycomprises determining expression levels of a cancer stem cell markerusing, for example, quantitative RT-PCR. In some embodiments, adiagnostic assay further comprises determining expression levels of acancer stem cell marker compared to a control tissue such as, forexample, normal epithelium.

Detection of a cancer stem cell marker expression can then be used toprovide a prognosis and select a therapy. A prognosis can be based onany known risk expression of a cancer stem cell marker indicates.Furthermore, detection of a cancer stem cell marker can be used toselect an appropriate therapy including, for example, treatment with anantagonist against the detected cancer stem cell marker. In certainembodiments, the antagonist is an antibody that specifically binds tothe extracellular domain of a cancer stem cell marker protein such as ahuman NOTCH receptor selected from the group consisting of NOTCH1,NOTCH2, NOTCH3 and NOTCH4.

In other embodiments of the present invention, diagnosis of a patient ismade by detection of a cancer stem cell gene signature as provided inU.S. patent application No. 60/690,003. In certain embodiments, apatient is screened for the presence of a tumor or benign adenoma orpolyps that indicate a pre-disposition to cancer. A biopsy from apatient is then analyzed for the presence of a cancer stem cell genesignature. In some embodiments, expression of a cancer stem cell genesignature is determined by polynucleotide expression such as, forexample, mRNA encoding the cancer stem cell gene signature. Thepolynucleotides of a cancer gene signature can be detected andquantified by any of a number of means well known in the art. In otherembodiments, expression of a cancer stem cell gene signature can bedetermined by detection of the corresponding polypeptides. Thepolypeptides can be detected and quantified by any of a number of meanswell known in the art.

Detection of a cancer stem cell gene signature can then be used toprovide a prognosis and select a treatment. A prognosis can be based onthe expression of any risk known at the time as reflected in the cancerstem cell gene signature. Furthermore, detection of a cancer stem cellgene signature can be used to select an appropriate therapy including,for example, treatment with an antagonist against a detected cancer stemcell marker. In some embodiments, the antagonist is an antibody thatspecifically binds to the extracellular domain of a cancer stem cellmarker protein such as NOTCH1.

In other embodiments of the present invention, patients screened for thepresence of colon adenomas or polyps are tested for allelic loss andsomatic mutations via a genetic test. In some embodiments the genetictest screens for loss or mutations in the Wnt pathway including, forexample, APC, Axin2 or beta-catenin. Notch signaling can play a role inmaintenance of the undifferentiated state of neoplastic cells activatedby unregulated Wnt signaling (van Es & Clevers, 2005, Trends in Mol.Med. 11:496-502), thus antagonists against the cancer stem cell markerNOTCH1 can be used as a treatment for Wnt-activated colon cancers. Insome embodiments, the antagonist is an antibody that specifically bindsto the extracellular domain of NOTCH1.

Cancer Stem Cell Marker Antagonists

In the context of the present invention, a suitable antagonist is anagent that can have one or more of the following effects, for example:interfere with the expression of a cancer stem cell marker; interferewith activation of a cancer stem cell signal transduction pathway by,for example, sterically inhibiting interactions between a cancer stemcell marker and its ligand, receptor or co-receptors; or bind to acancer stem cell marker and trigger cell death or inhibit cellproliferation.

In certain embodiments, antagonists against a cancer stem cell markeract extracellularly to act upon or inhibit the function of a cancer stemcell marker. In certain embodiments, an antagonist is a small moleculethat binds to the extracellular domain of a cancer stem cell markerprotein. In certain embodiments, an antagonist of a cancer stem cellmarker is proteinaceous. In some embodiments, proteinaceous antagonistsof a cancer stem cell marker are antibodies that specifically bind to anextracellular epitope of a cancer stem cell marker protein.Extracellular binding of an antagonist against a cancer stem cell markercan inhibit the signaling of a cancer stem cell marker protein byinhibiting intrinsic activation (e.g. kinase activity) of a cancer stemcell marker and/or by sterically inhibiting the interaction, forexample, of a cancer stem cell marker with its ligand, of a cancer stemcell marker with its receptor, of a cancer stem cell marker with aco-receptor, or of a cancer stem cell marker with the extracellularmatrix. Furthermore, extracellular binding of an antagonist against acancer stem cell marker can down-regulate cell-surface expression of acancer stem cell marker such as, for example, by internalization of acancer stem cell marker protein and/or decreasing cell surfacetrafficking of a cancer stem cell marker.

In some embodiments, antagonists against a cancer stem cell marker bindto a cancer stem cell marker and have one or more of the followingeffects: inhibit proliferation of tumor cells, trigger cell deathdirectly in tumor cells, or prevent metastasis of tumor cells. Incertain embodiments, antagonists of a cancer stem cell marker triggercell death via a conjugated toxin, chemotherapeutic agent, radioisotope,or other such agent. For example, an antibody against a cancer stem cellmarker is conjugated to a toxin that is activated in tumor cellsexpressing the cancer stem cell marker by protein internalization. Inother embodiments, antagonists of a cancer stem cell marker mediate celldeath of a cell expressing the cancer stem cell marker protein viaantibody-dependent cellular cytotoxicity (ADCC). ADCC involves celllysis by effector cells that recognize the Fc portion of an antibody.Many lymphocytes, monocytes, tissue macrophages, granulocytes andeosinophils, for example, have Fc receptors and can mediate cytolysis(Dillman, 1994, J. Clin. Oncol. 12:1497). In some embodiments, anantagonist of a cancer stem cell marker is an antibody that triggerscell death of cell expressing a cancer stem cell marker protein byactivating complement-dependent cytotoxicity (CDC). CDC involves bindingof serum complement to the Fc portion an antibody and subsequentactivation of the complement protein cascade, resulting in cell membranedamage and eventual cell death. Biological activity of antibodies isknown to be determined, to a large extent, by the constant domains or Fcregion of the antibody molecule (Uananue and Benacerraf, Textbook ofImmunology, 2nd Edition, Williams & Wilkins, p. 218 (1984)). Antibodiesof different classes and subclasses differ in this respect, as doantibodies of the same subclass but from different species. Of humanantibodies, IgM is the most efficient class of antibodies to bindcomplement, followed by IgG1, IgG3, and IgG2 whereas IgG4 appears quitedeficient in activating the complement cascade (Dilhman, 1994, J. Clin.Oncol. 12:1497; Jefferis et al., 1998, Immunol. Rev. 163:59-76).According to the present invention, antibodies of those classes havingthe desired biological activity are prepared.

The ability of any particular antibody against a cancer stem cell tomediate lysis of the target cell by complement activation and/or ADCCcan be assayed. The cells of interest are grown and labeled in vitro;the antibody is added to the cell culture in combination with eitherserum complement or immune cells which can be activated by the antigenantibody complexes. Cytolysis of the target cells is detected, forexample, by the release of label from the lysed cells. In fact,antibodies can be screened using the patient's own serum as a source ofcomplement and/or immune cells. The antibody that is capable ofactivating complement or mediating ADCC in the in vitro test can then beused therapeutically in that particular patient.

In other embodiments, antagonists of a cancer stem cell marker cantrigger cell death indirectly by inhibiting angiogenesis. Angiogenesisis the process by which new blood vessels form from pre-existing vesselsand is a fundamental process required for normal growth, for example,during embryonic development, wound healing and in response toovulation. Solid tumor growth larger than 1-2 mm² also requiresangiogenesis to supply nutrients and oxygen without which tumor cellsdie. Thus in certain embodiments, an antagonist of a cancer stem cellmarker targets vascular cells that express the cancer stem cell markerincluding, for example, endothelial cells, smooth muscle cells orcomponents of the extracellular matrix required for vascular assembly.In other embodiments, an antagonist of a cancer stem cell markerinhibits growth factor signaling required by vascular cell recruitment,assembly, maintenance or survival.

Antibodies

Recently the application of antibodies to target tumor cells has beendiscovered and used successfully against CD20 expressing B-cells innon-Hodgkin Lymphoma and HER2 and EGFR overexpressing breast cancers.Antibodies against growth factor receptors can inhibit growth factorreceptor function, inhibiting the growth of tumor cells as well asrendering these cells more susceptible to cytotoxic agents.Additionally, antibodies can mediate complement-dependent cytotoxicityor antibody-dependent cellular cytotoxicity to kill tumors expressing atarget antigen. Antibodies can also be directly conjugated to toxins orradioisotopes to mediate tumor cell killing. Furthermore, tumor survivaldepends on neo-vascularization, and targeting angiogenesis viaantibodies against VEGF has been used successfully to prolong patientsurvival.

The present invention provides isolated antibodies against a cancer stemcell marker. The antibody, or antibody fragment, can be any monoclonalor polyclonal antibody that specifically recognizes the described cancerstem cell marker. In some embodiments, the present invention providesmonoclonal antibodies, or fragments thereof, that specifically bind to acancer stem cell marker polypeptide described herein. In someembodiments, the monoclonal antibodies, or fragments thereof, arechimeric or humanized antibodies that specifically bind to theextracellular domain of a cancer stem cell marker polypeptide describedherein. In other embodiments, the monoclonal antibodies, or fragmentsthereof, are human antibodies that specifically bind to theextracellular domain of a cancer stem cell marker polypeptide describedherein.

The antibodies against a cancer stem cell marker find use in theexperimental, diagnostic and therapeutic methods described herein. Incertain embodiments, the antibodies of the present invention are used todetect the expression of a cancer stem cell marker protein in biologicalsamples such as, for example, a patient tissue biopsy, pleural effusion,or blood sample. Tissue biopsies can be sectioned and protein detectedusing, for example, immunofluorescence or immunohistochemistry.Alternatively, individual cells from a sample are isolated, and proteinexpression detected on fixed or live cells by FACS analysis.Furthermore, the antibodies can be used on protein arrays to detectexpression of a cancer stem cell marker, for example, on tumor cells, incell lysates, or in other protein samples. In other embodiments, theantibodies of the present invention are used to inhibit the growth oftumor cells by contacting the antibodies with tumor cells either invitro cell based assays or in vivo animal models. In still otherembodiments, the antibodies are used to treat cancer in a human patientby administering a therapeutically effective amount of an antibodyagainst a cancer stem cell marker.

Polyclonal antibodies can be prepared by any known method. Polyclonalantibodies are raised by immunizing an animal (e.g. a rabbit, rat,mouse, donkey, etc.) by multiple subcutaneous or intraperitonealinjections of the relevant antigen (a purified peptide fragment,full-length recombinant protein, fusion protein, etc.) optionallyconjugated to keyhole limpet hemocyanin (KLH), serum albumin, etc.diluted in sterile saline and combined with an adjuvant (e.g. Completeor Incomplete Freund's Adjuvant) to form a stable emulsion. Thepolyclonal antibody is then recovered from blood, ascites and the like,of an animal so immunized. Collected blood is clotted, and the serumdecanted, clarified by centrifugation, and assayed for antibody titer.The polyclonal antibodies can be purified from serum or ascitesaccording to standard methods in the art including affinitychromatography, ion-exchange chromatography, gel electrophoresis,dialysis, etc.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein (1975) Nature 256:495. Using thehybridoma method, a mouse, hamster, or other appropriate host animal, isimmunized as described above to elicit the production by lymphocytes ofantibodies that will specifically bind to an immunizing antigen.Alternatively, lymphocytes can be immunized in vitro. Followingimmunization, the lymphocytes are isolated and fused with a suitablemyeloma cell line using, for example, polyethylene glycol, to formhybridoma cells that can then be selected away from unfused lymphocytesand myeloma cells. Hybridomas that produce monoclonal antibodiesdirected specifically against a chosen antigen as determined byimmunoprecipitation, immunoblotting, or by an in vitro binding assaysuch as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay(ELISA) can then be propagated either in vitro culture using standardmethods (Goding, Monoclonal Antibodies: Principles and Practice,Academic Press, 1986) or in vivo as ascites tumors in an animal. Themonoclonal antibodies can then be purified from the culture medium orascites fluid as described for polyclonal antibodies above.

Alternatively monoclonal antibodies can also be made using recombinantDNA methods as described in U.S. Pat. No. 4,816,567. The polynucleotidesencoding a monoclonal antibody are isolated from mature B-cells orhybridoma cell, such as by RT-PCR using oligonucleotide primers thatspecifically amplify the genes encoding the heavy and light chains ofthe antibody, and their sequence is determined using conventionalprocedures. The isolated polynucleotides encoding the heavy and lightchains are then cloned into suitable expression vectors, which whentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, monoclonal antibodies aregenerated by the host cells. Also, recombinant monoclonal antibodies orfragments thereof of the desired species can be isolated from phagedisplay libraries as described (McCafferty et al., 1990, Nature,348:552-554; Clackson et al., 1991, Nature, 352:624-628; and Marks etal., 1991, J. Mol. Biol., 222:581-597).

The polynucleotide(s) encoding a monoclonal antibody can further bemodified in a number of different manners using recombinant DNAtechnology to generate alternative antibodies. In some embodiments, theconstant domains of the light and heavy chains of, for example, a mousemonoclonal antibody can be substituted 1) for those regions of, forexample, a human antibody to generate a chimeric antibody or 2) for anon-immunoglobulin polypeptide to generate a fusion antibody. In otherembodiments, the constant regions are truncated or removed to generatethe desired antibody fragment of a monoclonal antibody. Furthermore,site-directed or high-density mutagenesis of the variable region can beused to optimize specificity, affinity, etc. of a monoclonal antibody.

More generally, modified antibodies useful in the present invention maybe obtained or derived from any antibody. Further, the parent orprecursor antibody, or fragment thereof, used to generate the disclosedmodified antibodies may be murine, human, chimeric, humanized, non-humanprimate or primatized. In other embodiments the modified antibodies ofthe present invention can comprise single chain antibody constructs(such as that disclosed in U.S. Pat. No. 5,892,019, which isincorporated herein by reference) having altered constant domains asdescribed herein. Consequently, any of these types of antibodiesmodified in accordance with the teachings herein are compatible withthis invention.

According to the present invention, techniques can be adapted for theproduction of single-chain antibodies specific to a polypeptide of theinvention (see U.S. Pat. No. 4,946,778). In addition, methods can beadapted for the construction of Fab expression libraries (Huse, et al.,Science 246:1275-1281 (1989)) to allow rapid and effectiveidentification of monoclonal Fab fragments with the desired specificityfor NOTCH, or derivatives, fragments, analogs or homologs thereof.Antibody fragments that contain the idiotypes to a polypeptide of theinvention may be produced by techniques in the art including, but notlimited to: (a) an F(ab′)₂ fragment produced by pepsin digestion of anantibody molecule; (b) an Fab fragment generated by reducing thedisulfide bridges of an F(ab′)₂ fragment, (c) an Fab fragment generatedby the treatment of the antibody molecule with papain and a reducingagent, and (d) Fv fragments.

Bispecific antibodies are also within the scope of the invention.Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is foran antigenic polypeptide of the invention (NOTCH, or a fragmentthereof), while the second binding target is any other antigen, andadvantageously is a cell surface protein, or receptor or receptorsubunit.

Methods for making bispecific antibodies are known in the art.Traditionally the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain/light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature 305:537-539 (1983)). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatography.

Antibody variable domains with the desired binding specificities can befused to immunoglobulin constant domain sequences. The fusion is with animmunoglobulin heavy chain constant domain, comprising at least part ofthe hinge, CH2 and CH3 regions. The first heavy chain constant region(CH1) containing the site necessary for light chain binding can bepresent in at least one of the fusions. DNA encoding the immunoglobulinheavy chain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are co-transfected into asuitable host organism. Further details of generating bispecificantibodies can be found in Suresh et al., Methods in Enzymology 121:210(1986).

Bispecific antibodies can be prepared as full-length antibodies orantibody fragments. Techniques for generating bispecific antibodies fromantibody fragments have been described in the literature. For example,bispecific antibodies can be prepared using chemical linkage. Inaddition, Brennan et al., Science 229:81 (1985) describe a procedurewherein intact antibodies are proteolytically cleaved to generateF(ab′)₂ fragments.

Additionally, Fab′ fragments can be directly recovered from E. coli andchemically coupled to form bispecific antibodies (Shalaby et al., J.Exp. Med. 175:217-225 (1992)). These methods can be used in theproduction of a fully humanized bispecific antibody F(ab′)₂ molecule.

Antibodies with more than two valencies are also contemplated. Forexample, trispecific antibodies can be prepared (Tutt et al., J.Immunol. 147:60 (1991)).

Exemplary bispecific antibodies can bind to two different epitopes, atleast one of which originates in a polypeptide of the invention.Alternatively, an anti-antigenic arm of an immunoglobulin molecule canbe combined with an arm which binds to a triggering molecule on aleukocyte such as a T cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG so as to focus cellular defense mechanismsto the cell expressing the particular antigen. Bispecific antibodies canalso be used to direct cytotoxic agents to cells which express aparticular antigen. These antibodies possess an antigen-binding arm andan arm which binds a cytotoxic agent or a radionuclide chelator, such asEOTUBE, DPTA, DOTA, or TETA.

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune cells to unwanted cells (U.S. Pat. No. 4,676,980). It iscontemplated that the antibodies can be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins can be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

For the purposes of the present invention, it should be appreciated thatmodified antibodies can comprise any type of variable region thatprovides for the association of the antibody with the polypeptides ofNOTCH. In this regard, the variable region may comprise or be derivedfrom any type of mammal that can be induced to mount a humoral responseand generate immunoglobulins against the desired tumor associatedantigen. As such, the variable region of the modified antibodies can be,for example, of human, murine, non-human primate (e.g. cynomolgusmonkeys, macaques, etc.) or lupine origin. In some embodiments both thevariable and constant regions of the modified immunoglobulins are human.In other embodiments the variable regions of compatible antibodies(usually derived from a non-human source) can be engineered orspecifically tailored to improve the binding properties or reduce theimmunogenicity of the molecule. In this respect, variable regions usefulin the present invention can be humanized or otherwise altered throughthe inclusion of imported amino acid sequences.

In some embodiments, of the present invention the monoclonal antibodyagainst a cancer stem cell marker is a humanized antibody. Humanizedantibodies are antibodies that contain minimal sequences from non-human(e.g. murine) antibodies within the variable regions. Such antibodiesare used therapeutically to reduce antigenicity and HAMA (humananti-mouse antibody) responses when administered to a human subject. Inpractice, humanized antibodies are typically human antibodies withminimum to no non-human sequences. A human antibody is an antibodyproduced by a human or an antibody having an amino acid sequencecorresponding to an antibody produced by a human.

Humanized antibodies can be produced using various techniques known inthe art. An antibody can be humanized by substituting the CDR of a humanantibody with that of a non-human antibody (e.g. mouse, rat, rabbit,hamster, etc.) having the desired specificity, affinity, and capability(Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988,Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536).The humanized antibody can be further modified by the substitution ofadditional residue either in the Fv framework region and/or within thereplaced non-human residues to refine and optimize antibody specificity,affinity, and/or capability.

Human antibodies can be directly prepared using various techniques knownin the art. Immortalized human B lymphocytes immunized in vitro orisolated from an immunized individual that produce an antibody directedagainst a target antigen can be generated (See, for example, Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985); Boemer et al., 1991, J. Immunol., 147 (1):86-95; and U.S. Pat.No. 5,750,373). Also, the human antibody can be selected from a phagelibrary, where that phage library expresses human antibodies (Vaughan etal., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998, PNAS,95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Markset al., 1991, J. Mol. Biol., 222:581). Humanized antibodies can also bemade in transgenic mice containing human immunoglobulin loci that arecapable upon immunization of producing the full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Thisapproach is described in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;5,625,126; 5,633,425; and 5,661,016.

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array into such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann etal., Year in Immuno. 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825,5,591,669 (all of GenPharm); 5,545,807; and WO 97/17852.

Alternatively, phage display technology can be used to produce humanantibodies and antibody fragments in vitro, from immunoglobulin variable(V) domain gene repertoires from unimmunized donors. According to thistechnique, antibody V domain genes are cloned in-frame into either amajor or minor coat protein gene of a filamentous bacteriophage, such asM13 or fd, and displayed as functional antibody fragments on the surfaceof the phage particle. Because the filamentous particle contains asingle-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. Thus, the phagemimics some of the properties of the B-cell. Phage display can beperformed in a variety of formats. Several sources of V-gene segmentscan be used for phage display. A diverse array of anti-oxazoloneantibodies have been isolated from a small random combinatorial libraryof V genes derived from the spleens of immunized mice. A repertoire of Vgenes from unimmunized human donors can be constructed and antibodies toa diverse array of antigens (including self-antigens) can be isolated.

As discussed above, human antibodies may also be generated by in vitroactivated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

It will be appreciated that grafting the entire non-human variabledomains onto human constant regions will produce “classic” chimericantibodies. In the context of the present application the term “chimericantibodies” will be held to mean any antibody wherein the immunoreactiveregion or site is obtained or derived from a first species and theconstant region (which may be intact, partial or modified in accordancewith this invention) is obtained from a second species. In someembodiments, the antigen binding region or site will be from a non-humansource (e.g. mouse) and the constant region is human. While theimmunogenic specificity of the variable region is not generally affectedby its source, a human constant region is less likely to elicit animmune response from a human subject than would the constant region froma non-human source.

The variable domains in both the heavy and light chains are altered byat least partial replacement of one or more CDRs and, if necessary, bypartial framework region replacement and sequence changing. Although theCDRs may be derived from an antibody of the same class or even subclassas the antibody from which the framework regions are derived, it isenvisaged that the CDRs will be derived from an antibody of differentclass and preferably from an antibody from a different species. It mustbe emphasized that it may not be necessary to replace all of the CDRswith the complete CDRs from the donor variable region to transfer theantigen binding capacity of one variable domain to another. Rather, itmay only be necessary to transfer those residues that are necessary tomaintain the activity of the antigen binding site. Given theexplanations set forth in U.S. Pat. Nos. 5,585,089, 5,693,761 and5,693,762, it will be well within the art, either by carrying outroutine experimentation or by trial and error testing to obtain afunctional antibody with reduced immunogenicity.

Alterations to the variable region notwithstanding, it will beappreciated that the modified antibodies of this invention will compriseantibodies, or immunoreactive fragments thereof, in which at least afraction of one or more of the constant region domains has been deletedor otherwise altered so as to provide desired biochemicalcharacteristics such as increased tumor localization or reduced serumhalf-life when compared with an antibody of approximately the sameimmunogenicity comprising a native or unaltered constant region. In someembodiments, the constant region of the modified antibodies willcomprise a human constant region. Modifications to the constant regioncompatible with this invention comprise additions, deletions orsubstitutions of one or more amino acids in one or more domains. Thatis, the modified antibodies disclosed herein may comprise alterations ormodifications to one or more of the three heavy chain constant domains(CH1, CH2 or CH3) and/or to the light chain constant domain (CL). Insome embodiments of the invention modified constant regions wherein oneor more domains are partially or entirely deleted are contemplated. Inother embodiments the modified antibodies will comprise domain deletedconstructs or variants wherein the entire CH2 domain has been removed(ΔCH2 constructs). In still other embodiments the omitted constantregion domain will be replaced by a short amino acid spacer (e.g. 10residues) that provides some of the molecular flexibility typicallyimparted by the absent constant region.

Besides their configuration, it is known in the art that the constantregion mediates several effector functions. For example, binding of theC1 component of complement to antibodies activates the complementsystem. Activation of complement is important in the opsonisation andlysis of cell pathogens. The activation of complement also stimulatesthe inflammatory response and can also be involved in autoimmunehypersensitivity. Further, antibodies bind to cells via the Fc region,with a Fc receptor site on the antibody Fc region binding to a Fcreceptor (FcR) on a cell. There are a number of Fc receptors which arespecific for different classes of antibody, including IgG (gammareceptors), IgE (eta receptors), IgA (alpha receptors) and IgM (mureceptors). Binding of antibody to Fc receptors on cell surfacestriggers a number of important and diverse biological responsesincluding engulfment and destruction of antibody-coated particles,clearance of immune complexes, lysis of antibody-coated target cells bykiller cells (called antibody-dependent cell-mediated cytotoxicity, orADCC), release of inflammatory mediators, placental transfer and controlof immunoglobulin production. Although various Fc receptors and receptorsites have been studied to a certain extent, there is still much whichis unknown about their location, structure and functioning.

While not limiting the scope of the present invention, it is believedthat antibodies comprising constant regions modified as described hereinprovide for altered effector functions that, in turn, affect thebiological profile of the administered antibody. For example, thedeletion or inactivation (through point mutations or other means) of aconstant region domain may reduce Fc receptor binding of the circulatingmodified antibody thereby increasing tumor localization. In other casesit may be that constant region modifications, consistent with thisinvention, moderate complement binding and thus reduce the serum halflife and nonspecific association of a conjugated cytotoxin. Yet othermodifications of the constant region may be used to eliminate disulfidelinkages or oligosaccharide moieties that allow for enhancedlocalization due to increased antigen specificity or antibodyflexibility. Similarly, modifications to the constant region inaccordance with this invention may easily be made using well knownbiochemical or molecular engineering techniques.

It will be noted that the modified antibodies may be engineered to fusethe CH3 domain directly to the hinge region of the respective modifiedantibodies. In other constructs it may be desirable to provide a peptidespacer between the hinge region and the modified CH2 and/or CH3 domains.For example, compatible constructs could be expressed wherein the CH2domain has been deleted and the remaining CH3 domain (modified orunmodified) is joined to the hinge region with a 5-20 amino acid spacer.Such a spacer may be added, for instance, to ensure that the regulatoryelements of the constant domain remain free and accessible or that thehinge region remains flexible. However, it should be noted that aminoacid spacers may, in some cases, prove to be immunogenic and elicit anunwanted immune response against the construct. Accordingly, any spaceradded to the construct be relatively non-immunogenic or, even omittedaltogether if the desired biochemical qualities of the modifiedantibodies may be maintained.

Besides the deletion of whole constant region domains, it will beappreciated that the antibodies of the present invention may be providedby the partial deletion or substitution of a few or even a single aminoacid. For example, the mutation of a single amino acid in selected areasof the CH2 domain may be enough to substantially reduce Fc binding andthereby increase tumor localization. Similarly, it may be desirable tosimply delete that part of one or more constant region domains thatcontrol the effector function (e.g. complement CLQ binding) to bemodulated. Such partial deletions of the constant regions may improveselected characteristics of the antibody (serum half-life) while leavingother desirable functions associated with the subject constant regiondomain intact. Moreover, as alluded to above, the constant regions ofthe disclosed antibodies may be modified through the mutation orsubstitution of one or more amino acids that enhances the profile of theresulting construct. In this respect it may be possible to disrupt theactivity provided by a conserved binding site (e.g. Fc binding) whilesubstantially maintaining the configuration and immunogenic profile ofthe modified antibody. Yet other embodiments may comprise the additionof one or more amino acids to the constant region to enhance desirablecharacteristics such as effector function or provide for more cytotoxinor carbohydrate attachment. In such embodiments it can be desirable toinsert or replicate specific sequences derived from selected constantregion domains.

This invention also encompasses bispecific antibodies that specificallyrecognize a cancer stem cell marker. Bispecific antibodies areantibodies that are capable of specifically recognizing and binding atleast two different epitopes. The different epitopes can either bewithin the same molecule (e.g. the same cancer stem cell markerpolypeptide) or on different molecules such that both, for example, theantibodies can specifically recognize and bind a cancer stem cell markeras well as, for example, 1) an effector molecule on a leukocyte such asa T-cell receptor (e.g. CD3) or Fc receptor (e.g. CD64, CD32, or CD16)or 2) a cytotoxic agent as described in detail below. Bispecificantibodies can be intact antibodies or antibody fragments. Techniquesfor making bispecific antibodies are common in the art (Millstein etal., 1983, Nature 305:537-539; Brennan et al., 1985, Science 229:81;Suresh et al, 1986, Methods in Enzymol. 121:120; Traunecker et al.,1991, EMBO J. 10:3655-3659; Shalaby et al., 1992, J. Exp. Med.175:217-225; Kostelny et al., 1992, J. Immunol. 148:1547-1553; Gruber etal., 1994, J. Immunol. 152:5368; and U.S. Pat. No. 5,731,168).

In certain embodiments of the invention, it can be desirable to use anantibody fragment, rather than an intact antibody, to increase tumorpenetration, for example. Various techniques are known for theproduction of antibody fragments. Traditionally, these fragments arederived via proteolytic digestion of intact antibodies (for exampleMorimoto et al., 1993, Journal of Biochemical and Biophysical Methods24:107-117 and Brennan et al., 1985, Science, 229:81). However, thesefragments are now typically produced directly by recombinant host cellsas described above. Thus Fab, Fv, and scFv antibody fragments can all beexpressed in and secreted from E. coli or other host cells, thusallowing the production of large amounts of these fragments.Alternatively, such antibody fragments can be isolated from the antibodyphage libraries discussed above. The antibody fragment can also belinear antibodies as described in U.S. Pat. No. 5,641,870, for example,and can be monospecific or bispecific. Other techniques for theproduction of antibody fragments will be apparent.

It can further be desirable, especially in the case of antibodyfragments, to modify an antibody in order to increase its serumhalf-life. This can be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment by mutationof the appropriate region in the antibody fragment or by incorporatingthe epitope into a peptide tag that is then fused to the antibodyfragment at either end or in the middle (e.g., by DNA or peptidesynthesis).

The present invention further embraces variants and equivalents whichare substantially homologous to the chimeric, humanized and humanantibodies, or antibody fragments thereof, set forth herein. These cancontain, for example, conservative substitution mutations, i.e. thesubstitution of one or more amino acids by similar amino acids. Forexample, conservative substitution refers to the substitution of anamino acid with another within the same general class such as, forexample, one acidic amino acid with another acidic amino acid, one basicamino acid with another basic amino acid or one neutral amino acid byanother neutral amino acid. What is intended by a conservative aminoacid substitution is well known in the art.

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent. Cytotoxic agents includechemotherapeutic agents, growth inhibitory agents, toxins (e.g., anenzymatically active toxin of bacterial, fungal, plant, or animalorigin, or fragments thereof), radioactive isotopes (i.e., aradioconjugate), etc. Chemotherapeutic agents useful in the generationof such immunoconjugates include, for example, methotrexate, adriamicin,doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents. Enzymatically active toxins and fragments thereofthat can be used include diphtheria A chain, nonbinding active fragmentsof diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain,modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthinproteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),momordica charantia inhibitor, curcin, crotin, sapaonaria officinalisinhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, andthe tricothecenes. A variety of radionuclides are available for theproduction of radioconjugated antibodies including 212Bi, 131I, 131In,90Y, and 186Re. Conjugates of the antibody and cytotoxic agent are madeusing a variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Conjugates of an antibody and one ormore small molecule toxins, such as a calicheamicin, maytansinoids, atrichothene, and CC1065, and the derivatives of these toxins that havetoxin activity, can also be used.

Conjugate antibodies are composed of two covalently joined antibodies.Such antibodies have, for example, been proposed to target immune cellsto unwanted cells (U.S. Pat. No. 4,676,980). It is contemplated that theantibodies can be prepared in vitro using known methods in syntheticprotein chemistry, including those involving crosslinking agents. Forexample, immunotoxins can be constructed using a disulfide exchangereaction or by forming a thioether bond. Examples of suitable reagentsfor this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

In some embodiments the antibody of the invention contains human Fcregions that are modified to enhance effector function, for example,antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complementdependent cytotoxicity (CDC). This can be achieved by introducing one ormore amino acid substitutions in an Fc region of the antibody. Forexample, cysteine residue(s) can be introduced in the Fc region to allowinterchain disulfide bond formation in this region to improvecomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC) (Caron et al., 1992, J. Exp Med. 176:1191-1195;Shopes, 1992, Immunol. 148:2918-2922). Homodimeric antibodies withenhanced anti-tumor activity can also be prepared usingheterobifunctional cross-linkers as described in Wolff et al., 1993,Cancer Research 53:2560-2565. Alternatively, an antibody can beengineered which has dual Fc regions (Stevenson et al., 1989,Anti-Cancer Drug Design 3:219-230).

Regardless of how useful quantities are obtained, the antibodies of thepresent invention can be used in any one of a number of conjugated (i.e.an immunoconjugate) or unconjugated forms. Alternatively, the antibodiesof this invention can be used in a nonconjugated or “naked” form toharness the subject's natural defense mechanisms includingcomplement-dependent cytotoxicity (CDC) and antibody dependent cellulartoxicity (ADCC) to eliminate the malignant cells. In some embodiments,the antibodies can be conjugated to radioisotopes, such as ⁹⁰Y, ¹²⁵I,¹³¹I, ¹²³I, ¹¹¹In, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re and¹⁸⁸Re using anyone of a number of well known chelators or directlabeling. In other embodiments, the disclosed compositions can compriseantibodies coupled to drugs, prodrugs or biological response modifierssuch as methotrexate, adriamycin, and lymphokines such as interferon.Still other embodiments of the present invention comprise the use ofantibodies conjugated to specific biotoxins such as ricin or diptheriatoxin. In yet other embodiments the modified antibodies can be complexedwith other immunologically active ligands (e.g. antibodies or fragmentsthereof) wherein the resulting molecule binds to both the neoplasticcell and an effector cell such as a T cell. The selection of whichconjugated or unconjugated modified antibody to use will depend of thetype and stage of cancer, use of adjunct treatment (e.g., chemotherapyor external radiation) and patient condition. It will be appreciatedthat one could readily make such a selection in view of the teachingsherein.

Antibody Binding Assays

The antibodies of the present invention can be assayed forimmunospecific binding by any method known in the art. The immunoassayswhich can be used include, but are not limited to, competitive andnon-competitive assay systems using techniques such as BIAcore analysis,FACS analysis, immunofluorescence, immunocytochemistry, Western blots,radioimmunoassays, ELISA, “sandwich” immunoassays, immunoprecipitationassays, precipitin reactions, gel diffusion precipitin reactions,immunodiffusion assays, agglutination assays, complement-fixationassays, immunoradiometric assays, fluorescent immunoassays, and proteinA immunoassays. Such assays are routine and well known in the art (see,e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated byreference herein in its entirety).

In some embodiments, of the present invention the immunospecificity ofan antibody against a cancer stem cell marker is determined using ELISA.An ELISA assay comprises preparing antigen, coating wells of a 96 wellmicrotiter plate with antigen, adding the antibody against a cancer stemcell marker conjugated to a detectable compound such as an enzymaticsubstrate (e.g. horseradish peroxidase or alkaline phosphatase) to thewell, incubating for a period of time and detecting the presence of theantigen. Alternatively the antibody against a cancer stem cell marker isnot conjugated to a detectable compound, but instead a second conjugatedantibody that recognizes the antibody against a cancer stem cell markeris added to the well. Further, instead of coating the well with theantigen, the antibody against a cancer stem cell marker can be coated tothe well and a second antibody conjugated to a detectable compound canbe added following the addition of the antigen to the coated well. It isknown as to the parameters that can be modified to increase the signaldetected as well as other variations of ELISAs known in the art (seee.g. Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1).

The binding affinity of an antibody to a cancer stem cell marker antigenand the off-rate of an antibody-antigen interaction can be determined bycompetitive binding assays. One example of a competitive binding assayis a radioimmunoassay comprising the incubation of labeled antigen (e.g.3H or 125I), or fragment or variant thereof, with the antibody ofinterest in the presence of increasing amounts of unlabeled antigenfollowed by the detection of the antibody bound to the labeled antigen.The affinity of the antibody against a cancer stem cell marker and thebinding off-rates can be determined from the data by scatchard plotanalysis. In some embodiments, BIAcore kinetic analysis is used todetermine the binding on and off rates of antibodies against a cancerstem cell marker. BIAcore kinetic analysis comprises analyzing thebinding and dissociation of antibodies from chips with immobilizedcancer stem cell marker antigens on their surface.

Polynucleotides

The invention is directed to isolated polynucleotides encoding thepolypeptides of SEQ ID NOS: 2, 3, 5, 7-17 and 19, as well as thepolynucleotides of SEQ ID NOS:1, 4, 6 and 18. The polynucleotides of theinvention can be in the form of RNA or in the form of DNA, which DNAincludes cDNA, genomic DNA, and synthetic DNA. The DNA can bedouble-stranded or single-stranded, and if single stranded can be thecoding strand or non-coding (anti-sense) strand.

Thus, the term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequences for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequences.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs, andderivatives. The variant of the polynucleotide can be a naturallyoccurring allelic variant of the polynucleotide or a non-naturallyoccurring variant of the polynucleotide.

As hereinabove indicated, the polynucleotide can have a coding sequencewhich is a naturally occurring allelic variant of the coding sequence ofthe disclosed polypeptides. As known in the art, an allelic variant isan alternate form of a polynucleotide sequence which have asubstitution, deletion or addition of one or more nucleotides, whichdoes not substantially alter the function of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the codingsequence for the mature polypeptide can be fused in the same readingframe to a polynucleotide which aids in expression and secretion of apolypeptide from a host cell, for example, a leader sequence whichfunctions as a secretory sequence for controlling transport of apolypeptide from the cell. The polypeptide having a leader sequence is apreprotein and can have the leader sequence cleaved by the host cell toform the mature form of the polypeptide. The polynucleotides can alsoencode for a proprotein which is the mature protein plus additional 5′amino acid residues. A mature protein having a prosequence is aproprotein and is an inactive form of the protein. Once the prosequenceis cleaved an active mature protein remains.

Thus, for example, the polynucleotide of the present invention canencode for a mature protein, or for a protein having a prosequence orfor a protein having both a prosequence and presequence (leadersequence).

The polynucleotides of the present invention can also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence can be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencecan be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell 37:767 (1984)).

Further embodiments of the invention include isolated nucleic acidmolecules comprising a polynucleotide having a nucleotide sequence atleast 90% identical, 95% identical, and in some embodiments, at least96%, 97%, 98% or 99% identical to the disclosed sequences.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence can include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence. In other words, to obtain a polynucleotide having a nucleotidesequence at least 95% identical to a reference nucleotide sequence, upto 5% of the nucleotides in the reference sequence can be deleted orsubstituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence can be inserted intothe reference sequence. These mutations of the reference sequence canoccur at the amino- or carboxy-terminal positions of the referencenucleotide sequence or anywhere between those terminal positions,interspersed either individually among nucleotides in the referencesequence or in one or more contiguous groups within the referencesequence.

As a practical matter, whether any particular nucleic acid molecule isat least 95%, 96%, 97%, 98% or 99% identical to a reference sequence canbe determined conventionally using known computer programs such as theBestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). Bestfit uses the local homology algorithmof Smith and Waterman, Advances in Applied Mathematics 2: 482 489(1981), to find the best segment of homology between two sequences. Whenusing Bestfit or any other sequence alignment program to determinewhether a particular sequence is, for instance, 95% identical to areference sequence according to the present invention, the parametersare set, of course, such that the percentage of identity is calculatedover the full length of the reference nucleotide sequence and that gapsin homology of up to 5% of the total number of nucleotides in thereference sequence are allowed.

The polynucleotide variants can contain alterations in the codingregions, non-coding regions, or both. In some embodiments thepolynucleotide variants contain alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. In some embodiments,nucleotide variants are produced by silent substitutions due to thedegeneracy of the genetic code. Polynucleotide variants can be producedfor a variety of reasons, e.g., to optimize codon expression for aparticular host (change codons in the human mRNA to those preferred by abacterial host such as E. coli).

Polypeptides

The polypeptides of the present invention can be recombinantpolypeptides, natural polypeptides, or synthetic polypeptides having thesequence of SEQ ID NOS: 2, 3, 5, 7-17 and 19, as well as thepolypeptides encoded by the polynucleotides of SEQ ID NOS:1, 4, 6 and18.

It will be recognized in the art that some amino acid sequences of theinvention can be varied without significant effect of the structure orfunction of the protein. If such differences in sequence arecontemplated, it should be remembered that there will be critical areason the protein which determine activity.

Thus, the invention further includes variations of the polypeptideswhich show substantial activity or which include regions of NOTCHprotein such as the protein portions discussed herein. Such mutantsinclude deletions, insertions, inversions, repeats, and typesubstitutions. As indicated above, guidance concerning which amino acidchanges are likely to be phenotypically silent can be found in Bowie, J.U., et al., “Deciphering the Message in Protein Sequences: Tolerance toAmino Acid Substitutions,” Science 247:1306 1310 (1990).

Thus, the fragments, derivatives, or analogs of the polypeptides of theinvention can be: (I) one in which one or more of the amino acidresidues are substituted with a conserved or non-conserved amino acidresidue (often a conserved amino acid residue) and such substitutedamino acid residue can or can not be one encoded by the genetic code; or(ii) one in which one or more of the amino acid residues includes asubstituent group; or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol); or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, such asa leader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives, and analogs are deemed to be within the scope ofthe teachings herein.

Of particular interest are substitutions of charged amino acids withanother charged amino acid and with neutral or negatively charged aminoacids. The latter results in proteins with reduced positive charge toimprove the characteristics of the NOTCH protein. The prevention ofaggregation is highly desirable. Aggregation of proteins not onlyresults in a loss of activity but can also be problematic when preparingpharmaceutical formulations, because they can be immunogenic. (Pinckardet al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug CarrierSystems 10:307-377 (1993)).

As indicated, changes are typically of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Tables 1 and 2).

TABLE 1 Conservative Amino Acid Substitutions Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

TABLE 2 Amino Acid Substitutions Original Residue SubstitutionsExemplary Substitutions Ala (A) Val Val; Leu; Ile Arg (R) Lys Lys; Gln;Asn Asn (N) Gln Gln; His; Lys; Arg Asp (D) Glu Glu Cys (C) Ser Ser Gln(Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro Pro His (H) Arg Asn; Gln; Lys;Arg Ile (I) Leu Leu; Val; Met; Ala; Phe; norleucine Leu (L) Ilenorleucine; Ile; Val; Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn Met (M)Leu Leu; Phe; Ile Phe (F) Leu Leu; Val; Ile; Ala Pro (P) Gly Gly Ser (S)Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr Tyr (Y) Phe Trp; Phe; Thr; SerVal (V) Leu Ile; Leu; Met; Phe; Ala; norleucine

Of course, the number of amino acid substitutions made depends on manyfactors, including those described above. Generally speaking, the numberof substitutions for any given NOTCH polypeptide will not be more than50, 40, 30, 25, 20, 15, 10, 5 or 3.

The polypeptides and polynucleotides of the present invention areprovided in an isolated form, and at times are purified to homogeneity.

The polypeptides of the present invention include the polypeptides ofSEQ ID NOS:2, 3, 5, 7-17 and 19 as well as polypeptides which have atleast 90% similarity (at certain times at least 90% identity) to thepolypeptides of SEQ ID NOS:2, 3, 5, 7-17 and 19, and at least 95%similarity (at certain times at least 95% identity) to the polypeptidesof SEQ ID NOS: 2, 3, 5, 7-17 and 19, and still other embodiments, 96%,97%, 98%, or 99% similarity (at certain times 96%, 97%, 98%, or 99%identity) to the polypeptides of SEQ ID NOS: 2, 3, 5, 7-17 and 19. Asknown in the art “similarity” between two polypeptides is determined bycomparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention canbe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments can be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention can be used tosynthesize full-length polynucleotides of the present invention.

A fragment of the proteins of this invention is a portion or all of aprotein which is capable of binding to a cancer stem cell marker proteinor cancer stem cell protein binding partner (e.g. a receptor,co-receptor, ligand, or co-ligand). This fragment has a high affinityfor a cancer stem cell marker protein or cancer stem cell proteinbinding partner (e.g. a receptor, co-receptor, ligand, or co-ligand).Certain fragments of fusion proteins axe protein fragments comprising atleast part of the extracellular portion of a cancer stem cell markerprotein or cancer stem cell protein binding partner bound to at leastpart of a constant region of an immunoglobulin. The affinity istypically in the range of about 10-11 to 10-12 M, although the affinitycan vary considerably with fragments of different sizes, ranging from10-7 to 10-13 M. In some embodiments, the fragment is about 10-110 aminoacids in length and comprises the cancer stem cell marker protein ligandbinding site linked to at least part of a constant region of animmunoglobulin.

The polypeptides and analogs can be further modified to containadditional chemical moieties not normally part of the protein. Thosederivatized moieties can improve the solubility, the biological halflife or absorption of the protein. The moieties can also reduce oreliminate any desirable side effects of the proteins and the like. Anoverview for those moieties can be found in REMINGTON'S PHARMACEUTICALSCIENCES, 20th ed., Mack Publishing Co., Easton, Pa. (2000).

The isolated polypeptides described herein can be produced by anysuitable method known in the art. Such methods range from direct proteinsynthetic methods to constructing a DNA sequence encoding isolatedpolypeptide sequences and expressing those sequences in a suitabletransformed host. For example, cDNA can be obtained by screening a humancDNA library with a labeled DNA fragment encoding the polypeptide of SEQID NO: 1 and identifying positive clones by autoradiography. Furtherrounds of plaque purification and hybridization are performed usingconventional methods.

In some embodiments of a recombinant method, a DNA sequence isconstructed by isolating or synthesizing a DNA sequence encoding awild-type protein of interest. Optionally, the sequence can bemutagenized by site-specific mutagenesis to provide functional analogsthereof. See, e.g. Zoeller et al., Proc.-Nat. Acad. Sci. USA81:5662-5066 (1984) and U.S. Pat. No. 4,588,585. Another method ofconstructing a DNA sequence encoding a polypeptide of interest would beby chemical synthesis using an oligonucleotide synthesizer. Sucholigonucleotides can be designed based on the amino acid sequence of thedesired polypeptide and selecting those codons that are favored in thehost cell in which the recombinant polypeptide of interest will beproduced.

Standard methods can be applied to synthesize an isolated polynucleotidesequence encoding an isolated polypeptide of interest. For example, acomplete amino acid sequence can be used to construct a back-translatedgene. Further, a DNA oligomer containing a nucleotide sequence codingfor the particular isolated polypeptide can be synthesized. For example,several small oligonucleotides coding for portions of the desiredpolypeptide can be synthesized and then ligated. The individualoligonucleotides typically contain 5′ or 3′ overhangs for complementaryassembly.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the mutant DNA sequences encoding a particular isolatedpolypeptide of interest will be inserted into an expression vector andoperatively linked to an expression control sequence appropriate forexpression of the protein in a desired host. Proper assembly can beconfirmed by nucleotide sequencing, restriction mapping, and expressionof a biologically active polypeptide in a suitable host. As is wellknown in the art, in order to obtain high expression levels of atransfected gene in a host, the gene is operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host.

Recombinant expression vectors are used to amplify and express DNAencoding cancer stem cell marker polypeptide fusions. Recombinantexpression vectors are replicable DNA constructs which have synthetic orcDNA-derived DNA fragments encoding a cancer stem cell markerpolypeptide fusion or a bioequivalent analog operatively linked tosuitable transcriptional or translational regulatory elements derivedfrom mammalian, microbial, viral or insect genes. A transcriptional unitgenerally comprises an assembly of (1) a genetic element or elementshaving a regulatory role in gene expression, for example,transcriptional promoters or enhancers, (2) a structural or codingsequence which is transcribed into mRNA and translated into protein, and(3) appropriate transcription and translation initiation and terminationsequences, as described in detail below. Such regulatory elements caninclude an operator sequence to control transcription. The ability toreplicate in a host, usually conferred by an origin of replication, anda selection gene to facilitate recognition of transformants canadditionally be incorporated. DNA regions are operatively linked whenthey are functionally related to each other. For example, DNA for asignal peptide (secretory leader) is operatively linked to DNA for apolypeptide if it is expressed as a precursor which participates in thesecretion of the polypeptide; a promoter is operatively linked to acoding sequence if it controls the transcription of the sequence; or aribosome binding site is operatively linked to a coding sequence if itis positioned so as to permit translation. Generally, operatively linkedmeans contiguous and, in the case of secretory leaders, means contiguousand in reading frame. Structural elements intended for use in yeastexpression systems include a leader sequence enabling extracellularsecretion of translated protein by a host cell. Alternatively, whererecombinant protein is expressed without a leader or transport sequence,it can include an N-terminal methionine residue. This residue canoptionally be subsequently cleaved from the expressed recombinantprotein to provide a final product.

The choice of expression control sequence and expression vector willdepend upon the choice of host. A wide variety of expression host/vectorcombinations can be employed. Useful expression vectors for eukaryotichosts, include, for example, vectors comprising expression controlsequences from SV40, bovine papilloma virus, adenovims andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from Esherichia coli,including pCR 1, pBR322, pMB9 and their derivatives, wider host rangeplasmids, such as M13 and filamentous single-stranded DNA phages.

Suitable host cells for expression of a cancer stem cell marker proteininclude prokaryotes, yeast, insect or higher eukaryotic cells under thecontrol of appropriate promoters. Prokaryotes include gram negative orgram positive organisms, for example E. coli or bacilli. Highereukaryotic cells include established cell lines of mammalian origin asdescribed below. Cell-free translation systems could also be employed.Appropriate cloning and expression vectors for use with bacterial,fungal, yeast, and mammalian cellular hosts are described by Pouwels etal. (Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985), therelevant disclosure of which is hereby incorporated by reference.

Various mammalian or insect cell culture systems are also advantageouslyemployed to express recombinant protein. Expression of recombinantproteins in mammalian cells can be performed because such proteins aregenerally correctly folded, appropriately modified and completelyfunctional. Examples of suitable mammalian host cell lines include theCOS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175,1981), and other cell lines capable of expressing an appropriate vectorincluding, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO),HeLa and BHK cell lines. Mammalian expression vectors can comprisenontranscribed elements such as an origin of replication, a suitablepromoter and enhancer linked to the gene to be expressed, and other 5′or 3′ flanking nontranscribed sequences, and 5′ or 3′ nontranslatedsequences, such as necessary ribosome binding sites, a polyadenylationsite, splice donor and acceptor sites, and transcriptional terminationsequences. Baculovirus systems for production of heterologous proteinsin insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47(1988).

The proteins produced by a transformed host can be purified according toany suitable method. Such standard methods include chromatography (e.g.,ion exchange, affinity and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for protein purification. Affinity tags such as hexahistidine,maltose binding domain, influenza coat sequence andglutathione-S-transferase can be attached to the protein to allow easypurification by passage over an appropriate affinity column. Isolatedproteins can also be physically characterized using such techniques asproteolysis, nuclear magnetic resonance and x-ray crystallography.

For example, supernatants from systems which secrete recombinant proteininto culture media can be first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. Following the concentrationstep, the concentrate can be applied to a suitable purification matrix.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a cancer stem cell protein-Fc composition.Some or all of the foregoing purification steps, in variouscombinations, can also be employed to provide a homogeneous recombinantprotein.

Recombinant protein produced in bacterial culture is usually isolated byinitial extraction from cell pellets, followed by one or moreconcentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. High performance liquid chromatography (HPLC) canbe employed for final purification steps. Microbial cells employed inexpression of a recombinant protein can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

Inhibiting Tumor Cell Growth

The present invention also provides methods for inhibiting the growth oftumorigenic cells expressing a cancer stem cell marker using theantagonists of a cancer stem cell marker described herein. In someembodiments, the method of inhibiting the growth of tumorigenic cellsexpressing a cancer stem cell marker comprises contacting the cell withan antagonist against a cancer stem cell marker in vitro. For example,an immortalized cell line or a cancer cell line that expresses a cancerstem cell marker is cultured in medium to which is added an antagonistof the expressed cancer stem cell marker to inhibit cell growth.Alternatively tumor cells and/or tumor stem cells are isolated from apatient sample such as, for example, a tissue biopsy, pleural effusion,or blood sample and cultured in medium to which is added an antagonistof a cancer stem cell marker to inhibit cell growth. In someembodiments, the antagonist is an antibody that specifically recognizesan epitope of a cancer stem cell marker protein. For example, antibodiesagainst a cancer stem cell marker protein can be added to the culturemedium of isolated cancer stem cells to inhibit cell growth.

In some embodiments, the method of inhibiting the growth of tumorigeniccells expressing a cancer stem cell marker comprises contacting the cellwith an antagonist against a cancer stem cell marker in vivo. In certainembodiments, contacting a tumorigenic cell with an antagonist to acancer stem cell marker is undertaken in an animal model. For example,xenografts expressing a cancer stem cell marker are grown inimmunocompromised mice (e.g. NOD/SCID mice) that are administered anantagonist to a cancer stem cell marker to inhibit tumor growth.Alternatively, cancer stem cells that express a cancer stem cell markerare isolated from a patient sample such as, for example, a tissuebiopsy, pleural effusion, or blood sample and injected intoimmunocompromised mice that are then administered an antagonist againstthe cancer stem cell marker to inhibit tumor cell growth. In someembodiments, the antagonist of a cancer stem cell marker is administeredat the same time or shortly after introduction of tumorigenic cells intothe animal to prevent tumor growth. In other embodiments, the antagonistof a cancer stem cell marker is administered as a therapeutic after thetumorigenic cells have grown to a specified size. In some embodiments,the antagonist is a cancer stem cell marker protein fusion thatspecifically binds to a cancer stem cell marker protein or cancer stemcell marker binding protein (e.g. receptor, co-receptor, ligand, orco-ligand). In certain embodiments, the antagonist is an antibody thatspecifically recognizes an epitope of a cancer stem cell marker. Incertain embodiments, contacting a tumorigenic cell with an antagonist toa cancer stem cell is undertaken in a human patient diagnosed withcancer. In some embodiments, the antagonist is a cancer stem cell markerprotein fusion that specifically binds to a cancer stem cell markerprotein or cancer stem cell marker binding protein (e.g. receptor,co-receptor, ligand, or co-ligand). In other embodiments, the antagonistis an antibody that specifically recognizes an epitope of a cancer stemcell marker.

Pharmaceutical Compositions

The present invention further provides pharmaceutical compositionscomprising antagonists (e.g. antibodies) that target a cancer stem cellmarker. These pharmaceutical compositions find use in inhibiting tumorcell growth and treating cancer in human patients.

Formulations are prepared for storage and use by combining a purifiedantagonist (e.g. antibody) of the present invention with apharmaceutically acceptable carrier, excipient, and/or stabilizer as asterile lyophilized powder, aqueous solution, etc (Remington, TheScience and Practice of Pharmacy 20th Edition Mack Publishing, 2000).Suitable carriers, excipients, or stabilizers comprise nontoxic bufferssuch as phosphate, citrate, and other organic acids; salts such assodium chloride; antioxidants including ascorbic acid and methionine;preservatives (e.g. octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl orpropyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight polypeptides (less than about 10 aminoacid residues); proteins such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; carbohydrates such as monosaccharides, disaccharides,glucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN or polyethyleneglycol (PEG).

The pharmaceutical composition of the present invention can beadministered in any number of ways for either local or systemictreatment. Administration can be topical (such as to mucous membranesincluding vaginal and rectal delivery) such as transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders; pulmonary (e.g., by inhalation or insufflation of powdersor aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal); oral; or parenteral including intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial (e.g., intrathecal or intraventricular)administration.

The therapeutic formulation can be in unit dosage form. Suchformulations include tablets, pills, capsules, powders, granules,solutions or suspensions in water or non-aqueous media, or suppositoriesfor oral, parenteral, or rectal administration or for administration byinhalation. In solid compositions such as tablets the principal activeingredient is mixed with a pharmaceutical carrier. Conventionaltableting ingredients include corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, andother diluents (e.g. water) to form a solid preformulation compositioncontaining a homogeneous mixture of a compound of the present invention,or a non-toxic pharmaceutically acceptable salt thereof. The solidpreformulation composition is then subdivided into unit dosage forms ofthe type described above. The tablets, pills, etc of the novelcomposition can be coated or otherwise compounded to provide a dosageform affording the advantage of prolonged action. For example, thetablet or pill can comprise an inner composition covered by an outercomponent. Furthermore, the two components can be separated by anenteric layer that serves to resist disintegration and permits the innercomponent to pass intact through the stomach or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol and cellulose acetate.

Pharmaceutical formulations include antagonists of the present inventioncomplexed with liposomes (Epstein, et al., 1985, Proc. Natl. Acad. Sci.USA 82:3688; Hwang, et al., 1980, Proc. Natl. Acad. Sci. USA 77:4030;and U.S. Pat. Nos. 4,485,045 and 4,544,545). Liposomes with enhancedcirculation time are disclosed in U.S. Pat. No. 5,013,556. Someliposomes can be generated by the reverse phase evaporation with a lipidcomposition comprising phosphatidylcholine, cholesterol, andPEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes areextruded through filters of defined pore size to yield liposomes withthe desired diameter.

The antagonist can also be entrapped in microcapsules. Suchmicrocapsules are prepared, for example, by coacervation techniques orby interfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions as described in Remington, TheScience and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

In addition sustained-release preparations can be prepared. Suitableexamples of sustained-release preparations include semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles (e.g. films, ormicrocapsules). Examples of sustained-release matrices includepolyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) orpoly(vinylalcohol), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and 7 ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), sucrose acetateisobutyrate, and poly-D-(−)-3-hydroxybutyric acid.

Treatment with Antagonists

It is envisioned that the antagonists of the present invention can beused to treat various conditions characterized by expression and/orincreased responsiveness of cells to a cancer stem cell marker.Particularly it is envisioned that the antagonists (e.g. antibodies)against a cancer stem cell marker will be used to treat proliferativedisorders including but not limited to benign and malignant tumors ofthe kidney, liver, bladder, breast, stomach, ovary, colon, rectum,prostate, lung, vulva, thyroid, head and neck, brain (glioblastoma,astrocytoma, medulloblastoma, etc), blood and lymph (leukemias andlymphomas).

The antagonists are administered as an appropriate pharmaceuticalcomposition to a human patient according with known methods. Suitablemethod of administration include intravenous administration as a bolusor by continuous infusion over a period of time, by intramuscular,intraperitoneal, intravenous, intracerobrospinal, subcutaneous,intra-articular, intrasynovial, intrathecal, oral, topical, orinhalation routes.

In some embodiments, the treatment involves the combined administrationof an antagonist of the present invention and a chemotherapeutic agentor cocktail of multiple different chemotherapeutic agents. Treatmentwith an antagonist can occur prior to, concurrently with, or subsequentto administration of chemotherapies. Chemotherapies contemplated by theinvention include chemical substances or drugs which are known in theart and are commercially available, such as Doxorubicin, 5-Fluorouracil,Cytosine arabinoside (“Ara-C”), Cyclophosphamide, Thiotepa, Busulfan,Cytoxin, Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine andCarboplatin. Combined administration can include co-administration,either in a single pharmaceutical formulation or using separateformulations, or consecutive administration in either order butgenerally within a time period such that all active agents can exerttheir biological activities simultaneously. Preparation and dosingschedules for such chemotherapeutic agents can be used according tomanufacturers' instructions or as determined empirically. Preparationand dosing schedules for such chemotherapy are also described inChemotherapy Service Ed., M. C. Perry, Williams & Wilkins, Baltimore,Md. (1992).

In other embodiments, the treatment involves the combined administrationof an antagonist of the present invention and radiation therapy.Treatment with an antagonist can occur prior to, concurrently with, orsubsequent to administration of radiation therapy. Any dosing schedulesfor such radiation therapy can be used.

In other embodiments, the treatment can involve the combinedadministration of antibodies of the present invention with otherantibodies against additional tumor associated antigens including, butnot limited to, antibodies that bind to the EGF receptor (EGFR)(Erbitux®), the erbB2 receptor (HER2) (Herceptin®), and vascularendothelial growth factor (VEGF) (Avastin®). Furthermore, treatment caninclude administration of one or more cytokines, can be accompanied bysurgical removal of cancer cells or any other therapy deemed necessaryby a treating physician.

For the treatment of the disease, the appropriate dosage of anantagonist of the present invention depends on the type of disease to betreated, the severity and course of the disease, the responsiveness ofthe disease, whether the antagonist is administered for therapeutic orpreventative purposes, previous therapy, patient's clinical history, andso on all at the discretion of the treating physician. The antagonistcan be administered one time or over a series of treatments lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved (e.g. reduction in tumorsize). Optimal dosing schedules can be calculated from measurements ofdrug accumulation in the body of the patient and will vary depending onthe relative potency of an individual antagonist. The administeringphysician can easily determine optimum dosages, dosing methodologies andrepetition rates. In general, dosage is from 0.01 μg to 100 mg per kg ofbody weight, and can be given once or more daily, weekly, monthly oryearly. The treating physician can estimate repetition rates for dosingbased on measured residence times and concentrations of the drug inbodily fluids or tissues.

Kits

In yet other embodiments, the present invention provides kits that canbe used to perform the methods described herein. In some embodiments, akit comprises an antibody or antibodies specific for a cancer stem cellmarker, a purified antibody or antibodies, in one or more containers. Insome embodiments, a kit further comprises a substantially isolatedcancer stem cell marker polypeptide comprising an epitope that isspecifically immunoreactive with the antibody or antibodies included inthe kit, a control antibody that does not react with the cancer stemcell marker polypeptide, and/or a means for detecting the binding of anantibody to a cancer stem cell marker polypeptide (such as, for example,a fluorescent chromophore, an enzymatic substrate, a radioactivecompound or a luminescent compound conjugated to the antibody against acancer stem cell marker or to a second antibody that recognizes theantibody against a cancer stem cell marker). In other embodiments, a kitcomprises reagents specific for the detection of mRNA or cDNA (e.g.,oligonucleotide probes or primers) of one or more cancer stem cellmarker. In some embodiments, the kits contain all of the componentsnecessary and/or sufficient to perform a detection assay, including allcontrols, directions for performing assays, and any necessary softwarefor analysis and presentation of results.

A compartment kit includes any kit in which reagents are contained inseparate containers. Such containers include small glass containers,plastic containers or strips of plastic or paper. Such containers allowsone to efficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the antibodies or probes used in the methods,containers which contain wash reagents (such as phosphate bufferedsaline, Tris-buffers, etc.), and containers which contain the reagentsused to detect the bound antibody or probe. One will readily recognizethat the disclosed polynucleotides, polypeptides and antibodies of thepresent invention can be readily incorporated into one of theestablished kit formats which are well known in the art.

Screening Methods

In certain embodiments, the present invention provides a method ofidentifying a molecule that binds to a non-ligand binding region of anextracellular domain of a human NOTCH receptor and inhibits growth oftumor cells, the method comprising: i) incubating the molecule with thenon-ligand binding domain of the extracellular domain of the human Notchreceptor; ii) determining if the molecule binds to the non-ligandbinding region of the extracellular domain of the human Notch receptor;and iii) determining if the molecule inhibits growth of tumor cells.Molecules that specifically bind a non-ligand binding region of anextracellular domain of a human NOTCH receptor include, but are notlimited to, small organic molecules, polypeptides, and antibodies.

Screening can be performed using any suitable method known in the art.In certain embodiments, screening is performed in vitro. In someembodiments, cells expressing a non-ligand binding region of theextracellular domain of a human NOTCH receptor are incubated with alabeled molecule and specific binding of the labeled molecule to anon-ligand binding region of the extracellular domain of a human NOTCHreceptor is determined by FACS analysis. In some embodiments, anon-ligand binding region of the extracellular domain of a human NOTCHreceptor is expressed by phage display, and molecules that specificallybinding to a non-binding region of the extracellular domain of a humanNOTCH receptor are identified. Other suitable methods for identifyingmolecules that specifically bind to a non-ligand binding region of ahuman NOTCH receptor include, but are not limited to, ELISA; Western (orimmuno) blotting; and yeast-two-hybrid.

Molecules that specifically bind to a non-ligand binding region of anextracellular domain of a human NOTCH receptor are then tested forinhibition of tumor cell growth. Testing can be performed using anysuitable method known in the art. In certain embodiments, molecules thatspecifically bind to non-ligand binding region of the extracellulardomain of a human NOTCH receptor are tested for the ability to inhibittumor growth in vitro. In some embodiments, molecules that specificallybind a non-ligand binding region of the extracellular domain of a humanNOTCH receptor are incubated with tumor cells in culture andproliferation of tumor cells in the presence of a molecule thatspecifically binds a non-ligand binding region of the extracellulardomain of a human NOTCH receptor is determined and compared to tumorcells incubated with a non-binding control molecule. In certainembodiments, molecules that specifically bind to non ligand bindingregion of the extracellular domain of a human NOTCH receptor are testedfor the ability to inhibit tumor growth in vivo. In certain embodiments,molecules that specifically bind a non-ligand binding region of theextracellular domain of a human NOTCH receptor are injected into ananimal xenograft model and the growth of tumors in animals treated withmolecules that specifically bind to non-ligand binding region of theextracellular domain of a human NOTCH receptor is determined andcompared to animals treated with a non-binding control molecule.

EXAMPLES Example 1 Production of Antibodies

Antigen Production

Antibodies were generated against the non-ligand binding region ofNOTCH1 and NOTCH2.

In certain embodiments, recombinant polypeptide fragments of the humanNOTCH1 extracellular domain were generated as antigens for antibodyproduction. Standard recombinant DNA technology was used to isolatepolynucleotides encoding amino acids 1-220 of NOTCH1 (SEQ ID NO: 1) andamino acids 1427-1563 of NOTCH1 (SEQ ID NO: 18). These polynucleotideswere separately ligated in-frame N-terminal to a human Fc andhistidine-tag and cloned into a transfer plasmid vector for baculovirusmediated expression in insect cells. Standard transfection, infection,and cell culture protocols were used to produce recombinant insect cellsexpressing the corresponding NOTCH1 polypeptides corresponding to EGFrepeats 1-5 (SEQ ID NO: 2) and EGF repeats 10-15 (SEQ ID NO: 19)(O'Reilley et al., Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994)).

Cleavage of the endogenous NOTCH1 signal sequence was approximated usingcleavage prediction software SignalP 3.0 at between amino acid 18 and19, however the actual in vivo cleavage point can differ by a couple ofamino acids either direction. Thus NOTCH1 antigen protein correspondingto EGF repeats 1-5 comprises approximately amino acid 19 through aminoacid 220 (SEQ ID NO: 3). Antigen protein was purified from insect celllysates using protein A and Ni++-chelate affinity chromatography.Purified antigen protein was dialyzed against PBS (pH=7), concentratedto approximately 1 mg/ml, and sterile filtered in preparation forimmunization.

In certain embodiments, recombinant polypeptide fragments of the humanNOTCH2 extracellular domain were generated as antigens for antibodyproduction Standard recombinant DNA technology was used to isolate apolynucleotide encoding amino acids 1-493 of Notch2 (SEQ ID NO: 1). Thispolynucleotide was ligated in-frame N-terminal to either a human Fc-tagor histidine-tag and cloned into a transfer plasmid vector forbaculovirus mediated expression in insect cells. Standard transfection,infection, and cell culture protocols were used to produce recombinantinsect cells expressing the corresponding Notch2 polypeptide (O'Reilleyet al., Baculovirus expression vectors: A Laboratory Manual, Oxford:Oxford University Press (1994)).

Cleavage of the endogenous signal sequence of human Notch2 wasapproximated using cleavage prediction software SignalP 3.0, however theactual in vivo cleavage point can differ by a couple of amino acidseither direction. The predicated cleavage of Notch2 is between aminoacids 1 and 26, thus Notch2 antigen protein comprises approximatelyamino acid 27 through amino acid 493. Antigen protein was purified frominsect cell conditioned medium using Protein A and Ni⁺⁺-chelate affinitychromatography. Purified antigen protein was then dialyzed against PBS(pH=7), concentrated to approximately 1 mg/ml, and sterile filtered inpreparation for immunization.

Immunization

Mice (n=3) were immunized with purified NOTCH1 or NOTCH2 antigen protein(Antibody Solutions; Mountain View, Calif.) using standard techniques.Blood from individual mice was screened approximately 70 days afterinitial immunization for antigen recognition using ELISA and FACSanalysis (described in detail below). The two animals with the highestantibody titers were selected for final antigen boost after which spleencells were isolated for hybridoma production. Hybridoma cells wereplated at 1 cell per well in 96 well plates, and the supernatant fromeach well screened by ELISA and FACS analysis against antigen protein.Several hybridomas with high antibody titer were selected and scaled upin static flask culture. Antibodies were purified from the hybridomasupernatant using protein A or protein G agarose chromatography andantibodies were tested by FACS as described below. Several anti-NOTCH1antibodies were isolated including 13M57 (also referred to as 13M30)from animals immunized with NOTCH1 antigen corresponding to EGF repeats1-5 and 31M80, 31M103, 31M106, and 31M108 from animals immunized withNOTCH1 antigen corresponding to EGF repeats 10-15. The nucleotide andpredicted protein sequences of both the heavy chain (SEQ ID NO: 4-5) andlight chain (SEQ ID NO: 6-7) of antibody 13M57 were determined. A NOTCH2antibody, 59M07 (ATCC deposit no. PTA-10634; hybridoma deposited on Feb.4, 2010 at American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110-2209), was generated that specifically binds to EGFrepeat 2.

Epitope Mapping

To identify antibodies that recognize specific non-ligand bindingregions of the NOTCH receptor extracellular domains, epitope mapping waspreformed. In certain embodiments, mammalian expression plasmid vectorscomprising a CMV promoter upstream of polynucleotides that encodefragments of the extracellular NOTCH1 domain as Fc fusion proteins weregenerated using standard recombinant DNA technology. These fusionproteins included a series of NOTCH1 fragments containing a nestedseries of deletions of EGF domains 1-5 or 10-15. Recombinant proteinswere then expressed in cultured mammalian cells by transienttransfection. Twenty-four to 48 hours following transfection, cells wereharvested and cell lysate protein separated on non-reducing SDS-PAGEacrylamide gels for Western blotting using antibodies from miceimmunized with NOTCH1 antigen. As shown in FIGS. 1A and B monoclonalantibody 13M57 recognized an epitope contained within EGF repeat 4 (SEQID NO: 8) with a K_(D) of 0.2 nM. Monoclonal antibody 31M80 recognizedan epitope within EGF repeat 13 (FIG. 1C). Antibodies 31M103 (K_(D)=0.2nM), 31M106 (K_(D)=3 nM), and 31M108 (K_(D)=0.2 nM) also recognize anepitope within EGF13 of Notch1. All antibody affinities were determinedby BIAcore.

To identify specific epitopes within the extracellular domainsrecognized by an antibody against NOTCH1 the SPOTs system is used (SigmaGenosys, The Woodlands, Tex.). A series of 10-residue linear peptidesoverlapping by one amino acid and covering the entire NOTCH1extracellular domain are synthesized and covalently bound to a cellulosemembrane by the SPOT synthesis technique. The membrane is preincubatedfor 8 hours at room temperature with blocking buffer and hybridized withantibody overnight at 4° C. The membrane is then washed, incubated witha secondary antibody conjugated to horseradish peroxidase (HRP)(Amersham Bioscience, Piscataway, N.J.), re-washed, and visualized withsignal development solution containing 3-amino-9-ethylcarbazole.Specific epitopes recognized by an antibody are thus determined.

FACS Analysis

To select monoclonal antibodies produced by hybridoma clones thatrecognize native cell-surface NOTCH1 protein, FACs analysis was used. Tofacilitate the screening of cells by FACS, an isotype control mouseIgG1κ antibody and anti-NOTCH1 monoclonal antibody clones 13M57 and31M80 were conjugated to Alexa Fluor™ 647 (AF647) using Invitrogen kit#A-20186. The conjugation reaction resulted in approximately 0.1 mL ofAF647-labeled anti-Notch1 antibody at 1.0 mg/mL and AF647-labeledisotype control antibody at approximately 0.5 mg/mL. HEK293 cells weretransiently transfected with expression vectors encoding full lengthNOTCH1 and GFP. Twenty-four to 48-hours post-transfection cells werecollected in suspension and incubated on ice with either anti-NOTCH113M57 antibody, the corresponding anti-human NOTCH1 antisera, or controlIgG1 antibodies to detect background antibody binding. The cells werewashed and then sorted by FACS to identify antibody binding to cellsurface expressed NOTCH1. Anti-NOTCH1 antibody 13M57 recognizescell-surface NOTCH1 on HEK 293 cells (FIG. 2A). Similarly, anti-NOTCH1antibody 31M80 specifically recognizes HEK 293 cells transfected withNOTCH1 receptor but not untransfected cells, and this binding is blockedby increasing amounts of antigen protein containing NOTCH EGF repeats10-16 linked to human Fc (Ag31) but not antigen protein containing EGFrepeats 1-5 linked to Fc (Ag31) (FIG. 2B).

To determine if anti-NOTCH1 antibodies recognize NOTCH1 receptor proteinon tumor cells, dissociated tumor cells were analyzed by FACS.Dissociated breast tumor cells were incubated with anti-ESA, anti-CD44,and anti-NOTCH1 antibodies. Both 13M57 and 31M80 antibodies showedincreased binding to cells from two different breast tumors (PE13 andT3) compared to an isotype antibody control (FIGS. 2C, D). Furthermore,these breast tumor cells expressed high levels of ESA and CD44suggesting that both antibodies recognize NOTCH1 receptor on cancer stemcells (FIGS. 2C, D). Anti-NOTCH1 receptor antibody 31M80 also showedincreased binding to dissociated colon tumor cells from two differentpatients (FIG. 2E).

To determine whether NOTCH1 expression levels are directly associatedwith the tumorigenic activity of ESA+CD44+ cells, subpopulations of PE13cells were isolated by FACS based on 31M80-AF647 and CD44-PECy7 staining(FIG. 2C). Isolated cells are then injected into mice and theirtumorigenicity, the number of injected cells required for consistenttumor formation, is determined in comparison with ESA+CD44+NOTCH1^(low)and CD44− tumor cells.

Chimeric Antibodies

After monoclonal antibodies that specifically recognize a non-ligandbinding domain of a NOTCH receptor are identified, these antibodies aremodified to overcome the human anti-mouse antibody (HAMA) immuneresponse when rodent antibodies are used as therapeutics agents. Thevariable regions of the heavy-chain and light-chain of the selectedmonoclonal antibody are isolated by RT-PCR from hybridoma cells andligated in-frame to human IgG1 heavy-chain and kappa light chainconstant regions, respectively, in mammalian expression vectors.Alternatively a human Ig expression vector such as TCAE 5.3 is used thatcontains the human IgG1 heavy-chain and kappa light-chain constantregion genes on the same plasmid (Preston et al., 1998, Infection &Immunity 66:4137-42). Expression vectors encoding chimeric heavy- andlight-chains are then co-transfected into Chinese hamster ovary (CHO)cells for chimeric antibody production. Immunoreactivity and affinity ofchimeric antibodies are compared to parental murine antibodies by ELISAand FACS.

Humanized Antibodies

As chimeric antibody therapeutics are still frequently antigenic,producing a human anti-chimeric antibody (HACA) immune response,chimeric antibodies against a NOTCH1 receptor can require furtherhumanization. To generate humanized antibodies the three shorthypervariable sequences, or complementary determining regions (CDRs), ofthe chimeric antibody heavy- and light-chain variable domains describedabove are engineered using recombinant DNA technology into the variabledomain framework of a human heavy- and light-chain sequences,respectively, and then cloned into a mammalian expression vector forexpression in CHO cells. The immunoreactivity and affinity of thehumanized antibodies are compared to parental chimeric antibodies byELISA and FACS. Additionally, site-directed or high-density mutagenesisof the variable region can be used to optimize specificity, affinity,etc. of the humanized antibody.

Human Antibodies

In alternative embodiments, human antibodies that specifically recognizethe non-ligand, extracellular domain of a NOTCH receptor are isolatedusing phage display technology. A synthetic antibody library containinghuman antibody variable domains (MorphoSys, Munich, Germany) is screenedfor specific and high affinity recognition of a NOTCH receptor antigendescribed above. CDR cassettes in the library are specifically exchangedvia unique flanking restriction sites for antibody optimization.Optimized human variable regions are then cloned into an Ig expressionvector containing human IgG1 heavy-chain and kappa light-chain forexpression of human antibodies in CHO cells.

Example 2

In Vitro Assays to Evaluate Antibodies Against a NOTCH 1 Receptor

This example describes methods for in vitro assays to test the activityof antibodies generated against a NOTCH1 receptor on cell proliferationand cytotoxicity.

Ligand Binding

Antibodies against NOTCH1 were tested for their ability to block ligandbinding of the NOTCH receptor ligands DLL4 and JAGGED1 (JAG1). HEK 293cells stably transfected with a full-length NOTCH1 cDNA were incubatedwith either DLL4-Fc or JAG1-Fc in the presence of anti-NOTCH1 antibodies(13M57, 31M103, 31M106, or 31M108) or control anti-DLL4 (21M18) oranti-JAG1 (64M14) antibodies. Binding of Fc fusion proteins to cellsexpressing NOTCH1 was detected by PE-conjugated goat anti-Fc antibodyand flow cytometry.

Anti-NOTCH1 antibodies against EGF13 fail to effectively block ligandbinding (FIG. 3). Anti-DLL4 or anti-JAG1 antibody inhibition of DLL4 orJAG1 binding to the NOTCH1 receptor, respectively, was compared toinhibition by anti-NOTCH1 antibodies. Anti-NOTCH1 antibodies 31M103,31M106, and 31M108, all of which specifically bind to EGF13, onlypartially inhibit ligand binding. 31M108 showed between 50-75%inhibition compared to the ligand antibodies. Similarly, 31M103 and31M106 showed only between 25-50% inhibition.

Anti-NOTCH1 antibodies against EGF4 do no block ligand binding (FIG. 3).Again, anti-DLL4 or anti-JAG1 antibody inhibition of DLL4 or JAG1binding to NOTCH1 was compared to inhibition by the anti-NOTCH1antibodies 13M57, which specifically bind to EGF4. 13M57 showed noinhibition of ligand binding when compared to inhibition by anti-DLL4 oranti-JAG1 antibodies.

Proliferation Assay

Antibodies against NOTCH1 were tested for their effect on tumor cellgrowth in vitro using a BrdU based assay. Freshly dissociated,Lin-depleted breast tumor cells were cultured in low oxygen for between2-5 days. Cells were then cultured at 20,000 cells/well with 2.5 ug/mLor 5.0 ug/mL anti-NOTCH1 (13M57), control non-specific murine IgG, or noantibody for three days followed by 18 hours BrdU labeling. Allexperiments are performed with 5 replicates. The ability of anti-NOTCH1antibodies to inhibit cell proliferation compared to control antibodiesis shown in FIG. 4.

Complement-Dependent Cytotoxicity Assay

Cancer cell lines expressing a NOTCH1 receptor or, alternatively, cancerstem cells isolated from a patients sample passaged as a xenograft inimmunocompromised mice (described in detail below) are used to measurecomplement dependent cytotoxicity (CDC) mediated by an antibody againsta NOTCH1 receptor. Cells are suspended in 200 ul RPMI 1640 culturemedium supplemented with antibiotics and 5% FBS at 106 cells/ml.Suspended cells are then mixed with 200 ul serum or heat-inactivatedserum with antibodies against a NOTCH1 receptor or control antibodies intriplicate. Cell mixtures are incubated for 1 to 4 hours at 37° C. in 5%CO₂. Treated cells are then collected, resuspended in 100 ulFITC-labeled annexin V diluted in culture medium and incubated at roomtemperature for 10 min. One hundred ul of a propidium iodide solution(25 ug/ml) diluted in HBSS is added and incubated for 5 min at roomtemperature. Cells are collected, resuspended in culture medium andanalyzed by flow cytometry. Flow cytometry of FITC stained cellsprovides total cell counts, and propidium iodide uptake by dead cells asa percentage of total cell numbers is used to measure cell death in thepresence of serum and antibodies against a NOTCH1 compared toheat-inactivated serum and control antibodies. The ability ofanti-NOTCH1 antibodies to mediated complement-dependent cytotoxicity isthus determined.

Antibody-Dependent Cellular Cytotoxicity Assay

Cancer cell lines expressing a NOTCH1 receptor or, alternatively, cancerstem cells isolated from a patients sample passaged as a xenograft inimmunocompromised mice (described in detail below) are used to measureantibody dependent cellular cytotoxicity (ADCC) mediated by an antibodyagainst a NOTCH1 receptor. Cells are suspended in 200 ul phenol red-freeRPMI 1640 culture medium supplemented with antibiotics ad 5% FBS at 106cells/ml. Peripheral blood mononuclear cells (PBMCs) are isolated fromheparinized peripheral blood by Ficoll-Paque density gradientcentrifugation for use as effector cells. Target cells (T) are thenmixed with PBMC effector cells (E) at E/T ratios of 25:1, 10:1 and 5:1in 96-well plates in the presence of a NOTCH1 receptor or controlantibodies. Controls include incubation of target cells alone andeffector cells alone in the presence of antibody. Cell mixtures areincubated for 1 to 6 hours at 37° C. in 5% CO2. Released lactatedehydrogenase (LDH), a stable cytosolic enzyme released upon cell lysis,is then measured by a calorimetric assay (CytoTox96 Non-radioactiveCytotoxicity Assay; Promega; Madison, Wis.). Absorbance data at 490 nmare collected with a standard 96-well plate reader and backgroundcorrected. The percentage of specific cytotoxicity is calculatedaccording to the formula: % cytotoxicity=100×(experimental LDHrelease−effector spontaneous LDH release−target spontaneous LDHrelease)/(target maximal LDH release−target spontaneous LDH release).The ability of antibodies against a NOTCH1 receptor to mediated antibodydependent cellular cytotoxicity is thus determined.

Example 3

In Vivo Prevention of Tumor Growth Using Non-Ligand Binding RegionAnti-NOTCH Receptor Antibodies

This example describes the use of anti-NOTCH1 and anti-NOTCH2 receptorantibodies against a non-ligand binding region to prevent tumor growthin a xenograft model.

Tumor cells from a patient sample UM-PE13, OMP-C9, OMP-C8, and OMP-C6that have been passaged as a xenograft in mice were prepared forinjection into experimental animals. Tumor tissue was removed understerile conditions, cut up into small pieces, minced completely usingsterile blades, and single cell suspensions obtained by enzymaticdigestion and mechanical disruption. The resulting tumor pieces weremixed with ultra-pure collagenase III in culture medium (200-250 unitsof collagenase per mL) and incubated at 37° C. for 3-4 hours withpipetting up and down through a 10-mL pipette every 15-20 min. Digestedcells were filtered through a 45 ul nylon mesh, washed with RPMI/20%FBS, and washed twice with HBSS. Dissociated tumor cells were theninjected subcutaneously into NOD/SCID mice at 6-8 weeks to elicit tumorgrowth. For UM-PE13 breast tumor cells, 50,000 cells in 100 ul wereinjected into the right mammary fat pad (n=20) along with theimplantation of an estrogen pellet. For OMP-C9 colon tumor cells, 50,000cells in 100 ul were injected into the right flank region (n=20). ForOMP-C8 colon tumor cells, 10,000 cells in 100 ul were injected into theright flank area (n=10). For OMP-C6 colon tumor cells, 10,000 cells in100 ul were injected into the right flank (n=10).

In alternative embodiments, dissociated tumor cells are first sortedinto tumorigenic and non-tumorigenic cells based on cell surface markersbefore injection into experimental animals. Specifically, tumor cellsdissociated as described above are washed twice with Hepes bufferedsaline solution (HBSS) containing 2% heat-inactivated calf serum (HICS)and resuspended at 106 cells per 100 ul. Antibodies are added and thecells incubated for 20 min on ice followed by two washes with HBSS/2%HICS. Antibodies include anti-ESA (Biomeda, Foster City, Calif.),anti-CD44, anti-CD24, and Lineage markers anti-CD2, -CD3, -CD10, -CD16,-CD18, -CD31, -CD64, and -CD140b (collectively referred to as Lin;PharMingen, San Jose, Calif.). Antibodies are directly conjugated tofluorochromes to positively or negatively select cells expressing thesemarkers. Mouse cells are eliminated by selecting against H2Kd+ cells,and dead cells are eliminated by using the viability dye 7AAD. Flowcytometry is performed on a FACSVantage (Becton Dickinson, FranklinLakes, N.J.). Side scatter and forward scatter profiles are used toeliminate cell clumps. Isolated ESA+, CD44+, CD24−/low, Lin-tumorigeniccells are then injected subcutaneously into NOD/SCID mice to elicittumor growth.

Three days after tumor cell injection, antibody treatment was commenced.Each injected animal received 10 mg/kg anti-NOTCH1 antibodies or PBS asa control intraperitoneal (i.p.) two times per week for a total of 6 to8 weeks. Animals injected with UM-PE13 cells received injections intothe right upper mammary fat pad in addition to estrogen pelletinjections. Animals injected with OMP-C9, OMP-C8, or UM-C6 cellsreceived injections in the right lower quadrant of the abdomen. Tumorsize was assessed twice a week. Animals treated with anti-NOTCH1 13M57antibodies had significantly reduced PE-13 breast tumor cell growth(FIG. 5) and significantly reduced OMP-C9 colon tumor cell growth (FIG.6A) compared to PBS injected controls. Animals treated with anti-NOTCH113M57, 31M103, or 31M106 antibodies had significantly reduced OMP-C8colon tumor cell growth (FIG. 6B) compared to controls. Furtherexperimentation has indicated some variability in the effect ofanti-NOTCH1 13M57 antibodies in reducing OMP-C9 tumor cell growth inNOD/SCID mice.

Injection of PE-13 cells expressing luciferase under the control of astrong, constitutive promoter allows sensitive and accurate in vivodetection of tumor growth during the course of treatment. Animals areinjected with luciferin, which is converted by the luciferase enzyme toproduce light that can be imaged through the skin. Treatment with 13M30(13M57) antibodies significantly reduced growth of luciferase expressingPE-13 tumor cells compared to treatment with PBS or control antibodies(FIG. 7).

In certain embodiments, the anti-NOTCH2 antibody 59M07 was administeredat 10 mg/kg 2 days after C6 tumor cell injection. Antibody was giventwice weekly for a total of 48 days. Treatment with 59M07 significantlyreduced colon tumor cell growth (FIG. 8).

Example 4 Production of Antibodies Against NOTCH1, NOTCH2, NOTCH3, andNOTCH4 EGF Repeat 4

Identification of an antibody against the fourth EGF repeat of NOTCH1that reduces tumor growth in animals suggests the importance of thenon-ligand domain, and the fourth EGF repeat in particular, foreffective cancer therapies. To target the EGF repeat 4 in other Notchreceptor family members, antibodies against NOTCH2, NOTCH3, and NOTCH4EGF repeat 4 are produced and analyzed as described above foranti-NOTCH1 13M57. Specifically, mice are immunized with antigenscomprising the fourth EGF repeat of NOTCH2 (SEQ ID NO: 9); NOTCH3 (SEQID NO: 10), or NOTCH4 (SEQ ID NO: 11). Antibodies that recognizespecific Notch receptors as well as recognize different combinations ofthe four Notch receptors are identified using FACS analysis of HEK 293cells transfected with each Notch receptor as described in detail above.Antibodies that recognize the fourth EGF repeat of two Notch receptorfamily members are envisioned (e.g. antibodies that recognize the fourthEGF repeat of NOTCH1 and NOTCH2; NOTCH1 and NOTCH3; NOTCH1 and NOTCH4;NOTCH2 and NOTCH3; NOTCH2 and NOTCH4; or NOTCH3 and NOTCH4). Antibodiesthat recognize the fourth EGF repeat of three Notch receptor familymembers are envisioned (e.g. antibodies that recognize the fourth EGFrepeat of NOTCH1, NOTCH2, and NOTCH3; NOTCH1, NOTCH2, and NOTCH4; orNOTCH2, NOTCH3, and NOTCH4). And antibodies that recognize the fourthEGF repeat of four Notch receptor family members are envisioned (e.g.antibodies that recognize the fourth EGF repeat of NOTCH1, NOTCH2,NOTCH3 and NOTCH4).

Example 5 In Vivo Treatment of Tumors Using Anti-NOTCH1 ReceptorAntibodies

This example describes the use of anti-NOTCH1 receptor antibodies totreat cancer in a xenograft model.

Tumor cells from a patient sample (solid tumor biopsy or pleuraleffusion) that have been passaged as a xenograft in mice are preparedfor repassaging into experimental animals. Tumor tissue is removed, cutup into small pieces, minced completely using sterile blades, and singlecell suspensions obtained by enzymatic digestion and mechanicaldisruption. Dissociated tumor cells are then injected subcutaneouslyeither into the mammary fat pads, for breast tumors, or into the flank,for non-breast tumors, of NOD/SCID mice to elicit tumor growth.Alternatively, ESA+, CD44+, CD24−/low, Lin-tumorigenic tumor cells areisolated as described in detail above and injected.

Following tumor cell injection, animals are monitored for tumor growth.Once tumors reach an average size of approximately 150 to 200 mm,antibody treatment begins. Each animal receives 100 ug NOTCH1 receptorantibodies or control antibodies i.p. two to five times per week for atotal of 6 weeks. Tumor size is assessed twice a week during these 6weeks. The ability of NOTCH1 receptor antibodies to prevent furthertumor growth or to reduce tumor size compared to control antibodies isthus determined.

At the end point of antibody treatment, tumors are harvested for furtheranalysis. In some embodiments, a portion of the tumor is analyzed byimmunofluorescence to assess antibody penetration into the tumor andtumor response. A portion of each harvested tumor from anti-NOTCH1receptor treated and control antibody treated mice is fresh-frozen inliquid nitrogen, embedded in O.C.T., and cut on a cryostat as 10 umsections onto glass slides. Alternatively a portion of each tumor isformalin-fixed, paraffin-embedded, and cut on a microtome as 10 umsection onto glass slides. Sections are post-fixed and incubated withchromophore labeled antibodies that specifically recognize injectedantibodies to detect anti-NOTCH1 receptor or control antibodies presentin the tumor biopsy. Furthermore antibodies that detect different tumorand tumor recruited cell types such as, for example, anti-VE cadherin(CD144) or anti-PECAM-1 (CD31) antibodies to detect vascular endothelialcells, anti-smooth muscle alpha-actin antibodies detect vascular smoothmuscle cells, anti-Ki67 antibodies to detect proliferating cells, TUNELassays to detect dying cells, and anti-intracellular domain (ICD) Notchfragment antibodies to detect Notch signaling can be used to assessaffects of antibody treatment on angiogenesis, tumor growth and tumormorphology.

The effect of anti-NOTCH1 receptor antibody treatment on tumor cell geneexpression is also assessed. Total RNA is extracted from a portion ofeach harvested tumor from NOTCH1 antibody treated and control antibodytreated mice and used for quantitative RT-PCR. Expression levels ofNOTCH1, components of Notch signaling pathway including, as well asaddition cancer stem cell markers previously identified including, forexample, CD44 are analyzed relative to the house-keeping gene GAPDH asan internal control. Changes in tumor cell gene expression upon NOTCH1receptor antibody treatment are thus determined.

In addition, the effect of anti-NOTCH1 receptor antibody treatment onthe presence of cancer stem cells in a tumor is assessed. Tumor samplesfrom NOTCH1 versus control antibody treated mice are cut up into smallpieces, minced completely using sterile blades, and single cellsuspensions obtained by enzymatic digestion and mechanical disruption.Dissociated tumor cells are then analyzed by FACS analysis for thepresence of tumorigenic cancer stem cells based on ESA+, CD44+,CD24−/low, Lin-surface cell marker expression as described in detailabove.

The tumorigenicity of cells isolated based on ESA+, CD44+, CD24−/low,Lin-expression following anti-NOTCH1 antibody treatment can thenassessed. 5,000, 1,000, 500, and 100 isolated ESA+, CD44+, CD24−/low,Lin− cancer stem cells from NOTCH1 antibody treated versus controlantibody treated mice are re-injected subcutaneously into the mammaryfat pads of NOD/SCID mice. The tumorigenicity of cancer stem cells basedon the number of injected cells required for consistent tumor formationis thus determined.

Example 6 In Vivo Treatment of Tumors Using Combination TherapyAnti-NOTCH1 Receptor Antibodies and Chemotherapy

This example describes methods of treating cancer using combinationtherapy. Specifically anti-NOTCH1 antibodies in combination withchemotherapy treatment were used to treat both initial and establishedtumor growth in a xenograft model.

In certain embodiments, breast tumor cells were treated with acombination of anti-NOTCH1 antibodies and the chemotherapeutic,paclitaxel. Tumor cells from a patient sample UM-PE13 that have beenpassaged as a xenograft in mice were prepared for injection intoexperimental animals as described above. Dissociated UM-PE13 tumor cells(50,000 per animal) were injected subcutaneously into the right mammaryfat pad of NOD/SCID mice along with the implantation of an estrogenpellet. Tumors were allowed to grow for twenty-four days after which thetumors were measured and animals were split into four groups (n=10),each with an average tumor volume of 130 mm³. Each of the four groupswas treated as follows for 4 weeks: Group 1: paclitaxel; Group 2:paclitaxel; Group 3 paclitaxel+13M57; and Group 4: paclitaxel+13M57. Allinjections were administered IP, 2× per week. Paclitaxel was dosed at 15mg/kg and 13M57 was dosed at 30 mg/kg. Treatments with paclitaxel orpaclitaxel+13M57 were stopped at day 52 after the tumors had regressedand were undetectable. The animals were then treated as follows: Group1: PBS; Group 2: 13M57; Group 3: PBS; and Group 4: 13M57. Tumor volumewas checked once per week for the remainder of the experiment.

As shown in FIG. 9, concurrent combination treatment (paclitaxel+13M57)followed by continual treatment with anti-NOTCH1 13M57 antibodies hadthe greatest effect on tumor reoccurrence. Specifically, animals inGroup 4 that were treated with a combination of paclitaxel and 13M57 forfour weeks followed by continual treatment with 13M57 alone showedminimal tumor regrowth forty days following the termination ofpaclitaxel treatment (FIG. 9A). Only one animal in Group 4 showedappreciable tumor regrowth (FIG. 9B). Similarly, combination treatmentin which paclitaxel treatment was administered prior to 13M57 treatment(Group 2) also showed significant reduction in tumor regrowth comparedto animals treated with paclitaxel alone (Group 1), the latter of whichshowed tumor reoccurrence reaching tumor volumes near 100 mm³. Andfinally, combination therapy without continued treatment with anti-Notch13M57 antibodies (Group 3) also showed significant efficacy in reducingtumor regrowth compared to paclitaxel treatment alone (FIG. 9).

In certain embodiments, colon tumor cells were treated with acombination of anti-NOTCH1 antibodies and the chemotherapeutic,irinotecan. Tumor cells from the patient sample OMP-C8 passaged as axenograft in mice were prepared for injection into experimental animalsas described above. Dissociated OMP-C8 cells (20,000 cells per animal)were injected into NOD/SCID mice in the right lower quadrant of theabdomen. 2 days after cell injection, animals were treated either with7.5 mg/kg irinotecan alone (n=10) or with 10 mg/kg of the anti-NOTCH1antibody 13M57 plus 7.5 mg/kg irinotecan (n=10). Animals receivedtreatment two per week for up to 56 days, and tumor volume was assessedtwice weekly.

As shown in FIG. 10, concurrent combination treatment of irinotecan plus13M57 prevented colon tumor growth during treatment and also maintainedtumor cells in this non-proliferative state for up to thirty daysfollowing the cessation of treatment. Nearly all animals treated withirinotecan alone showed growth of tumors during the treatment phase,after which tumors continued to grow until termination of the experiment(FIG. 10A). In contrast, animals treated with 13M57 plus irinotecanshowed little to no tumor growth during combination treatment (FIG.10B). Furthermore, tumor cells remained quiescent post-treatment, withonly two animals showing tumor growth nearly thirty days (FIG. 10B).

In certain embodiments, colon tumor cells were treated with acombination of anti-NOTCH1 antibodies and the chemotherapeutic agent,oxaliplatin. Tumor cells from the patient sample OMP-C8 were preparedfor injection into experimental animals as described above. DissociatedOMP-C8 cells (20,000 cells per animal) were injected into NOD/SCID micein the right lower quadrant of the abdomen and were allowed to growuntil they reached an average tumor volume of 200 mm³. Animals (n=10 perexperimental group) were treated either with oxaliplatin (7.5 mg/kg), 10mg/kg 13M57, a combination of oxaliplatin and 13M57, or a controlantibody. Treatment was given twice weekly for 22 days; tumor volume wasassessed twice weekly.

As shown in FIG. 11, combination treatment was more effective ininhibiting established Tumor growth than oxaliplatin or 13M57 treatmentalone. Treatment with either anti-NOTCH1 13M57 or oxaliplatinsignificantly reduced tumor growth (p=0.04 vs. control), but combinationtreatment further reduced growth compared to treatment with either agentalone (p=0.03 vs. single agent) (FIG. 11).

Example 7

In Vivo Treatment of Tumors Using Combination Therapy

Anti-NOTCH1 and Anti-NOTCH2 Receptor Antibodies

This example describes methods of treating cancer using combinationantibody therapy. In certain embodiments, breast tumor cells weretreated with a combination of anti-NOTCH1 and anti-NOTCH2 antibodies.Tumor cells from the patient sample UM-PE13 expressing luciferase underthe control of a strong, constitutive promoter were prepared forinjection into experimental animals as described above. Dissociated PE13tumor cells (50,000 per animal) were injected subcutaneously into theright mammary fat pad of NOD/SCID mice along with an estrogen pellet.Two days following cell injection, animals were treated either with 10mg/kg anti-NOTCH1 31M108, 10 mg/kg 59M07 anti-NOTCH12, a combination ofanti-NOTCH1 and NOTCH2 antibodies, or a control antibody (n=8-10 foreach experimental group). Prior to imaging bioluminescence, animals wereinjected with luciferin; luciferin is converted by the luciferase enzymeto light that can be imaged through the skin. Animals were imaged andtumor volume was assessed twice weekly.

As shown in FIG. 12, treatment with a combination of NOTCH receptorantibodies has a significant effect on breast tumor cell growth.Treatment with 13M108 or 59M07 antibodies alone showed only a slightreduction in breast tumor growth (FIG. 12A). In contrast, combinationtreatment with anti-NOTCH1 and anti-NOTCH2 antibodies significantlyreduced growth of luciferase expressing PE13 tumor cells (FIG. 12A).Determination of total tumor volume showed a similar reduction in PE13breast tumor cells (FIG. 12B). Animals treated with 31M108 antibodiesshowed a significant reduction in total tumor volume compared to controltreated animals (p<0.05). Further reduction was observed in animalstreated with the antibody combination: treatment with NOTCH1 and NOTCH2antibodies reduced tumor growth significant compared to treatment witheither antibody alone (p<0.05) (FIG. 12B).

Example 8 Treatment of Human Cancer Using Anti-NOTCH1 ReceptorAntibodies

This example describes methods for treating cancer using antibodiesagainst a NOTCH1 receptor to target tumors comprising cancer stem cellsand/or tumor cells in which NOTCH1 receptor expression has beendetected.

The presence of cancer stem cell marker expression can first bedetermined from a tumor biopsy. Tumor cells from a biopsy from a patientdiagnosed with cancer are removed under sterile conditions. In someembodiments, the tissue biopsy is fresh-frozen in liquid nitrogen,embedded in O.C.T., and cut on a cryostat as 10 um sections onto glassslides. Alternatively the tissue biopsy is formalin-fixed,paraffin-embedded, and cut on a microtome as 10 um section onto glassslides. Sections are incubated with antibodies against a NOTCH1 receptorto detect protein expression. Additionally, the presence of cancer stemcells can be determined. Tissue biopsy samples are cut up into smallpieces, minced completely using sterile blades, and cells subject toenzymatic digestion and mechanical disruption to obtain a single cellsuspension. Dissociated tumor cells are then incubated with anti-ESA,-CD44, -CD24, -Lin, and -NOTCH1 antibodies to detect cancer stem cells,and the presence of ESA+, CD44+, CD24−/low, Lin−, NOTCH1+ tumor stemcells is determined by flow cytometry as described in detail above.

Cancer patients whose tumors are diagnosed as expressing a NOTCH1receptor are treated with anti-NOTCH1 receptor antibodies. Humanized orhuman monoclonal anti-NOTCH1 receptor antibodies generated as describedabove are purified and formulated with a suitable pharmaceutical carrierin PBS for injection. Patients are treated with the NOTCH1 antibodiesonce a week for at least 10 weeks, but in certain cases once a week forat least about 14 weeks. Each administration of the antibody should be apharmaceutically effective dose about 2 to about 100 mg/ml and incertain cases between about 5 to about 40 mg/ml. The antibody can beadministered prior to, concurrently with, or after standard radiotherapyregimens or chemotherapy regimens using one or more chemotherapeuticagent, such as oxaliplatin, fluorouracil, leucovorin, or streptozocin.Patients are monitored to determine whether such treatment has resultedin an anti-tumor response, for example, based on tumor regression,reduction in the incidences of new tumors, lower tumor antigenexpression, decreased numbers of cancer stem cells, or other means ofevaluating disease prognosis.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes for carrying outthe invention which are obvious to those in the relevant fields areintended to be within the scope of the following claims.

1. An isolated monoclonal antibody that specifically binds to anon-ligand binding region of the extracellular domain of human NOTCH2.2. The antibody of claim 1, wherein the antibody is a chimeric antibody.3. The antibody of claim 1, wherein the antibody is a humanizedantibody.
 4. The antibody of claim 1, wherein the antibody is a humanantibody.
 5. A hybridoma producing the antibody of claim
 1. 6. Theantibody of claim 1, wherein the non-ligand binding region comprisesepidermal growth factor (EGF) repeats 1-10.
 7. The antibody of claim 6,wherein the non-ligand binding region comprises EGF repeat
 4. 8. Theantibody of claim 6, wherein the non-ligand binding region comprises EGFrepeat
 2. 9. The antibody of claim 8, wherein the antibody is 59M07. 10.The antibody of claim 8, which is an antagonist of human NOTCH2.
 11. Theantibody of claim 8, which inhibits growth of colon tumor cells.
 12. Apharmaceutical composition comprising the antibody of claim
 8. 13. Theantibody of claim 1, wherein the non-ligand binding region comprisesepidermal growth factor (EGF) repeats 13-36.
 14. The antibody of claim13, wherein the non-ligand binding region comprises EGF repeat
 13. 15.The antibody of claim 1, which is an antagonist of human NOTCH2.
 16. Theantibody of claim 1, which inhibits the growth of colon tumor cells. 17.An isolated monoclonal antibody that competes with antibody 59M07 forspecific binding to a non-ligand binding region of the extracellulardomain of human NOTCH2.
 18. A monoclonal antibody that specificallybinds to the extracellular domain of human Notch 2, wherein the antibodybinds to epidermal growth factor (EGF) repeat
 10. 19. The antibody ofclaim 18, which is an antagonist of human NOTCH2.
 20. The antibody ofclaim 18, which inhibits growth of colon tumor cells.
 21. Apharmaceutical composition comprising the antibody of claim 18.